Tachykinin-like polypeptides and use thereof

ABSTRACT

The present invention relates to amino acid sequences for novel tachykinin polypeptides and base sequences encoding them, agonists and antagonists of such polypeptides, pharmaceutical compositions comprising them, and the like.

TECHNICAL FIELD

The present invention relates to novel tachykinin-like (poly)peptides(also referred to as “ATT” and “ATT polypeptides”) and precursorpolypeptides thereof (also referred to as “ATTα”, “ATTβ,” or “ATT#21F”),and to polynucleotides encoding them. The present invention also relatesto recombinant vectors containing such polynucleotides, transformantscontaining these vectors, and non-human transgenic animals having genescontaining such polynucleotides. The present invention furthermorerelates to methods for producing such polypeptides, antibodies againstsuch polypeptides, agonists and antagonists, and methods for identifyingthem. The present invention still furthermore relates to pharmaceuticalcompositions containing such polypeptides, polynucleotides, agonists,antagonists, or antibodies, as well as to methods for the treatmentand/or prevention of diseases, etc.

BACKGROUND ART

In all organisms, the coordinated regulation of events such as genesis,differentiation, growth, and homeostatic maintenance is dependent onintercellular and intertissue communication. In most cases, theseprocesses are mediated by protein factors. Many secretory factors(humoral factors) which have been discovered are involved in the immunesystem and hematopoietic system, for example, and are referred to ascytokines, which include lymphokines, monokines, interferons,colony-stimulating factors, tumor necrosis factors, and the like. Theirrelation to disease and their potential use as drugs continue to be thesubject of considerable research.

Humoral factors such as growth factors or peptide hormones produced byendocrine tissue also play an extremely important role in growth andhomeostatic maintenance, and their application as drugs is the subjectof considerable research.

For example, “tachykinins” is the generic term for a group ofphysiologically active peptides included in this category, which consistof about 10 amino acids and share in common the following carboxylterminal sequence motif:

Phe-Xaa-Gly-Leu-Met-NH₂

(the above sequence is an example in which the carboxyl group of the Cterminal methionine is amidated, where Xaa can be any amino acid).Substance P, neurokinin A, and neurokinin B are known mammaliantachykinins, and are widely distributed in the central nervous andperipheral nervous systems. These peptides are generally released by thenerves, and in most cases are known to bind to various receptors (NK-1,NK-2, and NK-3) present in the body, thereby producing various forms ofphysiological activity depending on their activation. Particularly interms of their pharmacological activity, it is clear from theliterature, including Pernow et al., Pharmacol. Rev., Vol. 35, pp.85-141 (1983), that proteins with the aforementioned carboxyl terminalstructure are shared. Examples of such activity are widely ranging, suchas smooth muscle contraction, decreased blood pressure, enhancedexocrine function, and stimulated vasopermeability. The release of suchpeptides is known to induce respiratory diseases such as respiratoryinflammation or bronchial contraction, and the release of histaminesfrom mast cells. Substance P, in particular, is believed to be involvedin the neurotransmission of pain, including pain associated withmigraines and arthritis. These peptides may also be involved ingastrointestinal disorders and diseases such as inflammatory boweldisease, as well as other diseases. In view of the great number ofclinical diseases characterized by tachykinin over-involvement, thedevelopment of tachykinin receptor antagonists may be useful incontrolling such clinical pathologies, and a number of peptide andnon-peptide tachykinin receptor antagonists have been developed thus far(Nippon Rinsho, Vol. 48, No. 5, p. 98-104 (1990), The FASEB Journal,Vol. 4, pp. 1606-1615 (1990), and Current Medical Chemistry, Vol. 6, pp.1375-1388 (1999)).

These protein and peptide factors, which are essential to organisms,have conventionally been found on the basis of their inherent biologicalactivity. Genes with high homology have then been discovered by means ofcloning techniques based on homology for known physiologically activeproteins. However, it is highly likely that, in addition to these knowngene groups, humoral functional molecules, which have not beenidentified by conventional techniques and whose existence thereforeremains unknown, also play a major physiological role in maintaining thehealth of higher organisms, especially mammals.

More recent research based on bioinformatics has attempted to makebiological, medical, and veterinary use of novel gene products, whichhave been discovered on the basis of DNA sequence data, through the aidof data processing techniques using computers (Trends in Biotechnology),Vol. 14, pp. 294-298 (1996). With recent achievements in the large-scalescreening of cDNA libraries, an enormous number of novel genes orcandidates have continued to be discovered through the compilation ofEST (expressed sequence tag) data. However, much of this sequence datais fragmentary and incomplete. A currently remaining, major issue isthat various existing cDNA-related public databases do not alwayscontain the complete expressed genes for various organisms. It can thusbe a daunting matter to search for completely novel and useful geneproducts in such databases. Meanwhile, structural analysis of all theDNA of a given organism, that is, the genome, has currently beencompleted for several bacteria and fungi (such as yeasts). Although suchresearch for the human genomes is expected to take several years, thenumber of human genes is estimated to be around a hundred thousand. Thegenes coding for many secretory proteins or secretory peptides haveactually been isolated already. However, that number cannot beunderstood to include all the genes of the genome. Despite the intensedesire to find novel useful products, the aforementioned data processingtechniques alone cannot be considered adequate for the elucidation ofsuch genes; proof must be based on more detailed biological or chemicalanalysis and experimentation.

DISCLOSURE OF THE INVENTION

An object of the present invention is thus to provide noveltachykinin-like polypeptides and their precursor polypeptides which areuseful for biological, medical, and veterinarian purposes, as well aspolynucleotides coding for them. Another object of the present inventionis to provide recombinant vectors containing such polynucleotides,transformants transformed with such vectors, and transgenic animalshaving such genes containing such polynucleotides. The invention is alsointended to provide a method for producing such polypeptides, antibodiesagainst such polypeptides, agonists or antagonists, and a method foridentifying them. Still another object of the present invention is toprovide pharmaceutical compositions containing such polypeptides,polynucleotides, antagonists, or antibodies, as well as methods fortreating or prevents diseases.

As a result of extensive research to resolve the above, the inventorssucceeded in finding cDNA with a novel base sequence which is expressedin human fetuses (skeletal muscle, lungs, heart, etc.) or the humanheart, human adipose tissue, human pituitary, or the like, anddiscovered that it coded for a precursor of a novel polypeptide ATT(atypical tachykinin). The inventors completed the present invention asa result of further research based on these findings.

That is, the present invention is intended to provide:

(1) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts, characterized bycomprising an amino acid sequence that is the same as or substantiallythe same as the amino acid sequence represented by SEQ ID NO. 17.

(2) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (1)above, characterized by comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 7;

(3) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (1)above, characterized by comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NOS. 3, 13, or 20;

(4) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (1)above, wherein the amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 17 is an amino acid sequence represented by SEQ ID NOS. 34, 35, 36,37, or 38;

(5) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts, characterized bycomprising an amino acid sequence that is the same as or substantiallythe same as the amino acid sequence represented by SEQ ID NO. 32;

(6) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (5)above, characterized by comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 22;

(7) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (5)above, wherein the amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 32 is an amino acid sequence represented by SEQ ID NOS. 23, 24, 25,26, 27, or 39;

(8) polypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (1) or(5) above, characterized by comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17 and an amino acid sequence that is the sameas or substantially the same as the amino acid sequence represented bySEQ ID NO. 32;

(9) amides, or their salts, of polypeptides or partial polypeptidesaccording to (1) or (5) above;

(10) polypeptides or partial peptides, or their salts according to (1)or (5), wherein the C terminal carboxyl group is amidated;

(11) polynucleotides comprising a polynucleotide coding for apolypeptide or partial peptide according to (1) or (5) above;

(12) polynucleotides according to (11) above, which are DNAs;

(13) polynucleotides according to (11) above, comprising a base sequencerepresented by SEQ ID NO. 4, 14, 21, 29, 30, 31, or 33;

(14) polynucleotides hybridizable with polynucleotides according to (11)above under stringent conditions;

(15) recombinant vectors comprising a polynucleotide according to (11)above;

(16) transformants transformed with vectors according to (15) above;

(17) a method for producing polypeptides or amides or esters thereof, ortheir salts, or partial peptides or amides or esters thereof, or theirsalts according to (1) or (5) above, characterized by culturingtransformants according to (16) above to produce and accumulatepolypeptides or partial peptides according to (1) or (5) above, andcollecting the polypeptides or partial peptides;

(18) non-human transgenic animals into which a polynucleotide accordingto (11) above or a vector according to (15) above is introduced;

(19) antibodies against polypeptides or amides or esters thereof, ortheir salts, or partial peptides or amides or esters thereof, or theirsalts according to (1) or (5) above;

(20) diagnostic agents comprising antibodies according to (19) above;

(21) a method for assaying polypeptides or partial peptides according to(1) or (5) above, characterized by the use of antibodies according to(19) above;

(22) antagonists against polypeptides or amides or esters thereof, ortheir salts, or partial peptides or amides or esters thereof, or theirsalts according to (1) or (5) above;

(23) agonists for polypeptides or amides or esters thereof, or theirsalts, or partial peptides or amides or esters thereof, or their saltsaccording to (1) or (5) above;

(24) agonists according to (23) above, which are agents for theprevention and/or treatment of hypertension;

(25) a method for screening antagonists according to (22) above oragonists according to (23) above, characterized by the use ofpolypeptides or amides or esters thereof, or their salts, or partialpeptides or amides or esters thereof, or their salts according to (1) or(5) above;

(26) a screening kit for antagonists according to (22) above or agonistsaccording to (23) above, characterized by comprising polypeptides oramides or esters thereof, or their salts, or partial peptides or amidesor esters thereof, or their salts according to (1) or (5) above;

(27) pharmaceutical compositions comprising polypeptides or amides oresters thereof, or their salts, or partial peptides or amides or estersthereof, or their salts according to (1) or (5) above;

(28) pharmaceutical compositions according to (27) above, which areagents for the prevention and/or treatment of hypertension;

(29) polynucleotides comprising a base sequence, or a portion thereof,that is complementary to a polynucleotide according to (11) above;

(30) a method for assaying the mRNA of polypeptides or amides or estersthereof, or their salts, or partial peptides or amides or estersthereof, or their salts according to (1) or (5) above, characterized bythe use of polynucleotides according to (11) above or portions thereof;

(31) the use of polypeptides or amides or esters thereof, or theirsalts, or partial peptides or amides or esters thereof, or their saltsaccording to (1) or (5) above for the production of pharmaceuticalcompositions comprising polypeptides or amides or esters thereof, ortheir salts, or partial peptides or amides or esters thereof, or theirsalts according to (1) or (5) above;

(32) a method for preventing and/or treating hypertension, characterizedby administering polypeptides or amides or esters thereof, or theirsalts, or partial peptides or amides or esters thereof, or their saltsaccording to (1) or (5) above to mammals;

(33) the use of agonists according to (23) above for the production ofdrugs comprising an agonist according to (23) above; and

(34) a method for preventing and/or treating hypertension, characterizedby administering an agonist according to (23) above to mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the base sequence and amino acid sequence of the openreading frame of ATTα precursor;

FIG. 2 illustrates the base sequence and amino acid sequence of the openreading frame of ATTβ precursor; and

FIG. 3 illustrates the contracting action of ATT(35-45) on a specimen ofguinea pig ileum.

The horizontal axis in the figure represents the dose (M) of ATT(35-45),and the vertical axis represents the rate of contraction relative to thecontraction of the guinea pig ileum specimens induced by 10-6 Macetylcholine.

BEST MODE FOR CARRYING OUT THE INVENTION

There are at least three types of the novel ATT polypeptide of thepresent invention. The precursor designated ATTα in the presentspecification comprises the 68 amino acids represented by SEQ ID NO. 3.The precursor designated ATTβ in the present Specification comprises the76 amino acids represented by SEQ ID NO. 13. ATTα and ATTβ share thesame sequence up to the glycine at position 67 from the N terminal ofthe amino acid sequence. Based on comparison with the base sequencedescribed below, these precursors are believed to be splice variantsfrom the same gene.

Another precursor of the novel ATT polypeptide in the present inventionis referred to as ATT#21F, comprising the 107 amino acids represented bySEQ ID NO. 20. ATT#21F shares the same sequence as ATTα and ATTβ up tothe glycine at position 67 from the N terminal of the amino acidsequence.

The novel mature ATT of the present invention (SEQ ID NO. 7) (sometimesreferred to below as ATT or ATT polypeptides) are produced upon theremoval of the signal sequence consisting of the 16 amino acidsrepresented by SEQ ID NO. 6 from the N terminal of ATTα, ATTβ, orATT#21F. At that time, the peptides may be subject to further limiteddegradation or modification. Polypeptides subject to such modificationor limited degradation (referred to below as ATTshort1 (SEQ ID NO. 17)and ATTshort2 (SEQ ID NO. 22)) are included within the scope of thepresent invention (this polypeptide and partial peptide are sometimesreferred to below as ATT or ATT polypeptide). The mature ATT producedfrom ATTα, ATTβ, and ATT#21F have the amino acid sequence represented bySEQ ID NO. 7, for example. Table 1 below compares the novel ATT sequenceof the present invention (designated ATT in Table 1 below) and sequencesof known peptides of the tachykinin family (in all examples, thecarboxyl groups of the C terminal is amidated).

TABLE 1 Comparison of sequences with known peptides of tachykinin family                                    RPKPQQFFGLM-NH₂ Substance P                                    HKTDSFVGLM-NH₂ Neurokinin A                                    DMHDFFVGLM-NH₂ Neurokinin BTVAGDGGEEQTLSTEAETWEGAGPSIQLQLQEVKTGKASQFFGLM-NH₂ ATT                                    TGKASQFFGLM-NH2 ATT-short

The aforementioned motif (FFGLM-NH₂) is present in the carboxyl terminalof the novel ATT of the present invention. Based on the sequencehomology, the polypeptides of the present invention can be concluded tohave physiological activity related to the tachykinins. The ATT of thepresent invention are believing to action and functions related to atleast substance P, neurokinin A, and neurokinin B.

Furthermore, ATT#21F of the present invention (SEQ ID NO. 20) not onlyhas the aforementioned motif represented by FFGLM-NH₂, but also has acarboxyl terminal sequence motif represented by Phe-Xaa-Gly-Leu-Met-NH₂(specifically, Phe-Gln-Gly-Leu-Met-NH₂, where Xaa is any amino acid),which is not known in extant naturally occurring proteins and peptides.This physiologically active peptide precursor has a function, which hasbeen completely unknown so far.

As used in the present specification, “polypeptides of the invention” isan expression used in a sense encompassing the aforementioned ATTα,ATTβ, ATT, ATT#21, ATTshort1, and ATTshort2. “Polypeptides of theinvention” is also an expression used in a sense encompassing amides ofthe polypeptides of the invention, esters of polypeptides of theinvention, and salts of polypeptides of the invention.

Polypeptides of the present invention and polynucleotides (such as DNA)coding for polypeptides of the present invention may be labeled bymethod that are well known per se. Specific examples includeradioisotope-labeled types, fluorescent-labeled types (such as withfluorescein), and biotinylated types or enzyme-labeled types.

Examples of salts of polypeptides of the present invention includepharmaceutically acceptable salts with acids or bases, and especiallypharmaceutically acceptable acid salts. Examples of such salts includesalts of inorganic acids (such as hydrochloric acid, phosphoric acid,hydrobromic acid, and sulfuric acid), and salts of organic acids (suchas acetic acid, formic acid, propionic acid, fumaric acid, maleic acid,succinic acid, tartaric acid, citric acid, malic acid, oxalic acid,benzoic acid, methanesulfonic acid, and benzenesulfonic acid).

“Polypeptides of the invention” are described in further detail below.

Polypeptides of the present invention may be synthetic polypeptides, orpolypeptides derived from cells (such as spleen cells, nerve cells, gliacells, pancreatic β cells, marrow cells, mesangial cells, Langerhans'cells, epidermal cells, epithelial cells, endothelial cells,fibroblasts, fibrocytes, myocytes, adipose cells, immunocytes (such asmacrophages, T cells, B cells, natural killer cells, mast cells,neutrophils, basophils, eosinophils, and monocytes), megakaryocytes,synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts,mammary gland cells, liver cells, and interstitial cells, or theircorresponding precursor cells, stem cells, cancer cells, and the like)or cells of the blood cell system, or any tissue in which such cells arepresent, such as the brain, regions of the brain (such as the olfactorybulb, amygdaloid nucleus, basal ganglia, hippocampus, thalamus,hypothalamus, subthalamic nucleus, cerebral cortex, medulla oblongata,cerebellum, occipital lobe, frontal lobe, temporal lobes, putamen,caudate nucleus, cerebral gland, and substantia nigra), spine, pituitarygland, stomach, pancreas, kidneys, liver, gonad, thyroid, gall bladder,bone marrow, adrenal gland, skin, muscle, lungs, gastrointestinal tract(such as the large intestine and small intestine), blood vessels, heart,thymus, spleen, submandibular gland, peripheral blood, peripheral bloodcells, prostate gland, testes, ovaries, placenta, uterus, bone, joints,and skeletal muscle (and particularly the brain and regions of thebrain) of humans or mammals (such as guinea pigs, rats, mice, rabbits,pigs, sheep, cows, and monkeys).

In the polypeptides of the invention, the left end is the N terminal(amino terminal) and the right end is the C terminal (carboxylterminal), according to the usual practice for describing peptides. Inthe polypeptides of the present invention, including polypeptides withan amino acid sequence represented by SEQ ID No. 17 or 32, theC-terminal is usually a carboxyl group (—COOH) or a carboxylate (—COO⁻),but the C-terminal may also be an amide (—CONH₂) or an ester (—COOR).

As used herein, R in such esters includes C₁ to C₆ alkyl such as methyl,ethyl, n-propyl, isopropyl, and n-butyl, C₃ to C₈ cycloalkyl such ascyclopentyl and cyclohexyl, C₆ to C₁₂ aryl such as phenyl andα-naphthyl, phenyl C₁ to C₂ alkyl such as benzyl or phenethyl,α-naphthyl-C₁ to C₂ alkyl such as α-naphthylmethyl, and other such C₇ toC₁₄ aralkyl, as well as pivaloyloxymethyl groups and the like which arecommonly used as esters for oral purposes.

In cases where the polypeptides of the present invention have carboxylgroups (or carboxylates) other than at the C terminal, polypeptides inwhich such carboxyl groups have been converted to amides or esters areincluded in the polypeptides of the present invention. Esters with suchC terminals may be used, for example, as esters in these cases.

The polypeptides of the present invention include such polypeptides inwhich the amino groups of the N terminal methionine residues areprotected with protective groups (such as formyl, acetyl or other suchC₂ to C₆ alkanoyl groups or similar C₁ to C₆ acyl groups), those inwhich the glutamyl produced upon in vivo cleavage of the N-terminal sideis converted to pyroglutamate, those in which substituents (such as —OH,—SH, amino groups, imidazole groups, indole groups, and guanidinegroups) on the side chains of the amino acids in the molecule areprotected with suitable protective groups (such as formyl, acetyl orother such C₂ to C₆ alkanoyl groups or similar C₁ to C₆ acyl groups),and conjugated proteins such as what are referred to as glycoproteinscomprising sugar linkages.

Detailed Description of Polypeptides of the Invention 1

Examples of polypeptides of the invention include:

(1) polypeptides comprising an amino acid sequence that is the same asor substantially the same as the amino acid sequence represented by SEQID NO. 17;

(2) polypeptides comprising an amino acid sequence that is the same asor substantially the same as the amino acid sequence represented by SEQID NO. 32;

Detailed Description of Polypeptides of the Invention 1-(1)

The amino acid sequence represented by SEQ ID NO. 17 is the amino acidsequence (Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met) represented bypositions 35 (Thr) to 45 (Met) from the N terminal of the aforementionedATT (SEQ ID NO. 7) (the peptide comprising this sequence is sometimesreferred to below as ATTshort1).

“Polypeptides comprising an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 17” can comprise any other amino acid sequence, provided that theyalso comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 17. When the amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 17 corresponds to the C terminal amino acid sequence of thepolypeptide, the C terminal carboxyl group of the polypeptide ispreferably amidated, or more specifically, the C terminal is representedby Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met-NH₂.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 17 include amino acidsequences with about at least 80%, more preferably about at least 90%,even more preferably about at least 95%, and still more preferably aboutat least 98% homology with the amino acid sequence represented by SEQ IDNO. 17.

As used in the present specification, homology refers to the extent ofthe match between two nucleotide sequences or two amino acid sequencesas expressed in terms of a percentage. Computers are generally used tosearch homology, using the well-known Smith-Waterman algorithm and theFASTA or BLAST program, or the like.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 17 include:

(i) amino acid sequences in which 1 or 2 or more (preferably 1 to about5, and more preferably 1 to about 3, and even more preferably 1 or 2) ofthe amino acids in the amino acid sequence represented by SEQ ID NO. 17have been deleted;

(ii) an amino acid sequence in which 1 or 2 or more (preferably 1 toabout 5, and more preferably 1 to about 3, and even more preferably 1 or2) amino acids have been added to the amino acid sequence represented bySEQ ID NO. 17;

(iii) an amino acid sequence in which 1 or 2 or more (preferably 1 toabout 5, and more preferably 1 to about 3, and even more preferably 1 or2) amino acids in the amino acid sequence represented by SEQ ID NO. 17have been substituted with other amino acids;

(iv) amino acid sequences in which the amino acid sequences (i) through(iii) above are combined;

(v) amino acid sequences in which 1 to about 3, and preferably 1 or 2amino acids other than those at positions 7 (Phe), 9 (Gly), 10 (Leu),and 11 (Met) from the N terminal in the amino acid sequence representedby SEQ ID NO. 17 have been deleted;

(vi) amino acid sequences in which 1 to about 3, and preferably 1 or 2amino acids other than those at positions 7 (Phe), 9 (Gly), 10 (Leu),and 11 (Met) from the N terminal in the amino acid sequence representedby SEQ ID NO. 17 have been substituted with other amino acids; and

(vii) amino acid sequences in which amino acid sequences (v) to (vi)above are combined.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 17 include proteins which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 17, and which have substantially the same activity as the amino acidsequence represented by SEQ ID NO. 17.

Examples of substantially the same activity include receptor-bindingactivity, signal-transduction activity, the hypotensive activitydescribed in Example 1 below, and the smooth muscle contracting activitydescribed in Example 2 below. “Substantially the same” means that theactivity is the same in terms of characteristics. As such, thereceptor-binding activity, signal-transduction activity, or the likeshould be the same (for example, about 0.01 to 100-fold, preferablyabout 0.5 to 20-fold, and more preferably about 0.5 to 2-fold), althoughthe quantitative factors such as the extent of the activity or thepolypeptide molecular weight may vary.

Activity such as the receptor-binding activity or signal-transductionactivity can be assayed in accordance with methods that are well knownper se, such as the screening method described below.

Specific examples of polypeptides with an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17 include the polypeptides described in“Detailed Description of Polypeptides of the Invention 2” and “DetailedDescription of Polypeptides of the Invention 5” below, but specificpreferred examples include:

(i) polypeptides consisting of an amino acid sequence represented by SEQID NO. 17;

(ii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 34;

(iii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 35;

(iv) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 36;

(v) polypeptides consisting of an amino acid sequence represented by SEQID NO. 37;

(vi) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 38; and

(vii) polypeptides in which the C terminal carboxyl groups of thepolypeptides in (i) through (vi) above are amidated.

Detailed Description of Polypeptides of the Invention 1-(2)

The amino acid sequence represented by SEQ ID NO. 32 is the amino acidsequence Phe-Xaa-Gly-Leu-Leu (where Xaa can be any amino acid) whichincludes the amino acid sequence from positions 85 (Phe) to 89 (Leu)from the N terminal (Phe-Gln-Gly-Leu-Leu: SEQ ID NO. 39) of theaforementioned ATT#21F (SEQ ID NO. 20).

Examples of amino acids represented by Xaa include glycine, alanine,valine, leucine, isoleucine, serine, threonine, cysteine, methionine,aspartic acid, glutamic acid, lysine, arginine, phenylalanine, tyrosine,histidine, tryptophan, asparagines, glutamine, and proline (same below).

“Polypeptides comprising an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 32” can comprise any other amino acid sequence, provided that theyalso comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 32. When the amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 32 corresponds to the C terminal amino acid sequence of thepolypeptide, the C terminal carboxyl group of the polypeptide ispreferably amidated. More specifically, the C terminal is represented byPhe-Xaa-Gly-Leu-Leu-NH₂ (where Xaa can be any amino acid).

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 32 include polypeptides which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 32, and which have substantially the same activity as the amino acidsequence represented by SEQ ID NO. 32.

Examples of substantially the same activity include receptor-bindingactivity, signal-transduction activity, the hypotensive activitydescribed in Example 1 below, and the smooth muscle contracting activitydescribed in Example 2 below. “Substantially the same” means that theactivity is the same in terms of characteristics. As such, thereceptor-binding activity, signal-transduction activity, or the likeshould be the same (for example, about 0.01 to 100-fold, preferablyabout 0.5 to 20-fold, and more preferably about 0.5 to 2-fold), althoughthe quantitative factors such as the extent of the activity or thepolypeptide molecular weight may vary.

Activity such as the receptor-binding activity or signal-transductionactivity can be assayed in accordance with methods that are well knownper se, such as the screening method described below.

Specific examples of polypeptides with an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 32 include the polypeptides described in“Detailed Description of Polypeptides of the Invention 3,” “DetailedDescription of Polypeptides of the Invention 4,” and “DetailedDescription of Polypeptides of the Invention 5” below, but specificpreferred examples include:

(i) polypeptides consisting of an amino acid sequence represented by SEQID NO. 32;

(ii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 39; and

(iii) polypeptides in which the C terminal carboxyl groups of thepolypeptides in (i) through (iii) above are amidated.

Detailed Description of Polypeptides of the Invention 2

More specific examples of “polypeptides with an amino acid sequence thatis the same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17” include:

(iii) polypeptides with an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 7; and

(iv) polypeptides with an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 3, 13, or 20.

Detailed Description of Polypeptides of the Invention 2-(1)

Polypeptides comprising an amino acid sequence represented by SEQ ID NO.7 mean the aforementioned ATT.

“Polypeptides comprising an amino acid sequence represented by SEQ IDNO. 7” can comprise any other amino acid sequence, provided that theyalso comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 7. When the amino acid sequence that is the same as or substantiallythe same as the amino acid sequence represented by SEQ ID NO. 7corresponds to the C terminal amino acid sequence of the polypeptide,the C terminal carboxyl group of the polypeptide is preferably amidated,or more specifically, the C terminal is represented byPhe-Xaa-Gly-Leu-Met-NH₂ (where Xaa can be any amino acid).

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 7 include polypeptides which have an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID 17, and which have an amino acid sequence with about at least80%, more preferably about at least 90%, even more preferably about atleast 95%, and still more preferably about at least 98% homology withthe amino acid sequence represented by SEQ ID NO. 7.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 7 include:

(i) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) of theamino acids in the amino acid sequence represented by SEQ ID NO. 7 havebeen deleted;

(ii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids have been added to the amino acid sequence represented bySEQ ID NO. 7;

(iii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids in the amino acid sequence represented by SEQ ID NO. 7have been substituted with other amino acids;

(iv) amino acid sequences in which amino acid sequences (i) through(iii) above are combined;

(v) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids from among amino acids (residues) other than those at positions 41(Phe), 43 (Gly), 44 (Leu), and 45 (Met) from the N terminal in the aminoacid sequence represented by SEQ ID NO. 7 have been deleted;

(vi) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids from among amino acids (residues) other than those at positions 41(Phe), 43 (Gly), 44 (Leu), and 45 (Met) from the N terminal in the aminoacid sequence represented by SEQ ID NO. 7 have been substituted withother amino acids; and

(vii) amino acid sequences in which amino acid sequences (v) to (vi)above are combined.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 7 include polypeptides which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 7, and which have substantially the same activity as the amino acidsequence represented by SEQ ID NO. 7.

Examples of substantially the same activity include receptor-bindingactivity, signal-transduction activity, the hypotensive activitydescribed in Example 1 below, and the smooth muscle contracting activitydescribed in Example 2 below. “Substantially the same” means that theactivity is the same in terms of characteristics. As such, thereceptor-binding activity, signal-transduction activity, or the likeshould be the same (for example, about 0.01 to 100-fold, preferablyabout 0.5 to 20-fold, and more preferably about 0.5 to 2-fold), althoughthe quantitative factors such as the extent of the activity or thepolypeptide molecular weight may vary.

Activity such as the receptor-binding activity or signal-transductionactivity can be assayed in accordance with methods that are well knownper se, such as the screening method described below.

Specific examples of polypeptides with an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 7 include the polypeptides described in“Detailed Description of Polypeptides of the Invention 2-(2)” below, butspecific preferred examples include:

(i) polypeptides consisting of an amino acid sequence represented by SEQID NO. 7; and

(ii) polypeptides in which the C terminal carboxyl groups ofpolypeptides consisting of the amino acid sequence represented by SEQ IDNO. 7 are amidated.

Detailed Description of Polypeptides of the Invention 2-(2)

Polypeptides comprising an amino acid sequence represented by SEQ ID NO.3 mean the aforementioned ATTα, Polypeptides comprising an amino acidsequence represented by SEQ ID NO. 13 mean the aforementioned ATTβ, andPolypeptides comprising an amino acid sequence represented by SEQ ID NO.20 mean the aforementioned ATT#21F.

“Polypeptides comprising an amino acid sequence represented by SEQ IDNOS. 3, 13, or 20” can comprise any other amino acid sequence, providedthat they also comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNOS. 3, 13, or 20.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequences represented by SEQ IDNO. 3, 13, or 20 include polypeptides which have an amino acid sequencethat is the same as or substantially the same as the amino acid sequencerepresented by SEQ ID 17, and which have an amino acid sequence withabout at least 60%, more preferably about at least 70%, even morepreferably about at least 80%, and still more preferably about at least90% homology with the amino acid sequences represented by SEQ ID NO. 3,13, or 20.

Examples of amino acid sequences that are substantially the same as theamino acid sequences represented by SEQ ID NOS. 3, 13, or 20 include:

(i) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) of theamino acids in the amino acid sequence represented by SEQ ID NOS. 3, 13,or 20 have been deleted;

(ii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids have been added to the amino acid sequence represented bySEQ ID NOS. 3, 13, or 20;

(iii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids in the amino acid sequence represented by SEQ ID NOS. 3,13, or 20 have been substituted with other amino acids;

(iv) amino acid sequences in which amino acid sequences (i) through(iii) above are combined;

(v) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids from among amino acids (residues) other than those at positions 57(Phe), 59 (Gly), 60 (Leu), and 61 (Met) from the N terminal in the aminoacid sequences represented by SEQ ID NOS. 3, 13, or 20 have beendeleted;

(vi) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 17, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids from among amino acids (residues) other than those at positions 57(Phe), 59 (Gly), 60 (Leu), and 61 (Met) from the N terminal in the aminoacid sequences represented by SEQ ID NOS. 3, 13, or 20 have beensubstituted with other amino acids; and

(vii) amino acid sequences in which amino acid sequences (v) to (vi)above are combined.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequences represented by SEQ IDNOS. 3, 13, or 20 include polypeptides which have an amino acid sequencethat is substantially the same as the amino acid sequences representedby SEQ ID NOS. 3, 13, or 20, and which have substantially the sameactivity as the amino acid sequences represented by SEQ ID NOS. 3, 13,or 20.

Examples of substantially the same activity include receptor-bindingactivity and signal-transduction activity. “Substantially the same”means that the activity is the same in terms of characteristics.However, because polypeptides which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNOS. 3, 13, or 20 may also be obtained in the form of ATT, ATTshort1,and ATTshort2 precursor polypeptides, they need not necessarily have thephysiological activity of ATT, ATTshort1, and ATTshort2 (such as thehypotensive activity described in Example 1 below or the smooth musclecontracting activity described in Example 2 below).

Specific preferred examples of polypeptides with an amino acid sequencethat is the same as or substantially the same as the amino acidsequences represented by SEQ ID NOS. 3, 13, or 20 include:

(i) polypeptides consisting of an amino acid sequence represented by SEQID NO. 3;

(ii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 13; and

(iii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 20.

Detailed Description of Polypeptides of the Invention 3

More specific examples of “polypeptides with an amino acid sequence thatis the same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 32” include:

(5) polypeptides with an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 22.

The amino acid sequence represented by SEQ ID NO. 22 is the amino acidsequence from positions 76 (Lys) through 89 (Leu)(Lys-Lys-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu) (thepolypeptide consisting of this sequence is sometimes referred to belowas “ATTshort2”) from the N terminal of ATT#21F (SEQ ID NO. 20).

“Polypeptides comprising an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 22” can comprise any other amino acid sequence, provided that theyalso comprises an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 22. When the amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 22 corresponds to the C terminal amino acid sequence of thepolypeptide, the C terminal carboxyl group of the polypeptide ispreferably amidated.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 22 include amino acidsequences with about at least 80%, more preferably about at least 90%,even more preferably about at least 95%, and still more preferably aboutat least 98% homology with the amino acid sequence represented by SEQ IDNO. 22.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 22 include:

(i) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 32, in which 1 or 2 or more (preferably 1 to about 5, morepreferably 1 to about 3, and even more preferably 1 or 2) of the aminoacids in the amino acid sequence represented by SEQ ID NO. 22 have beendeleted;

(ii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 32, in which 1 or 2 or more (preferably 1 toabout 5, more preferably 1 to about 3, and even more preferably 1 or 2)amino acids have been added to the amino acid sequence represented bySEQ ID NO. 22;

(iii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 32, in which 1 or 2 or more (preferably 1 toabout 5, more preferably 1 to about 3, and even more preferably 1 or 2)amino acids in the amino acid sequence represented by SEQ ID NO. 22 havebeen substituted with other amino acids;

(iv) amino acid sequences in which amino acid sequences (i) through(iii) above are combined;

(v) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 32, in which 1 to about 3, and preferably 1 or 2 aminoacids (residues) other than those at positions 10 (Phe), 12 (Gly), 13(Leu), and 14 (Leu) from the N terminal in the amino acid sequencerepresented by SEQ ID NO. 22 have been deleted;

(vi) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 32, in which 1 to about 3, and preferably 1 or 2 aminoacids (residues) amino acids (residues) other than those at positions 10(Phe), 12 (Gly), 13 (Leu), and 14 (Leu) from the N terminal in the aminoacid sequence represented by SEQ ID NO. 22 have been substituted withother amino acids; and

(vii) amino acid sequences in which amino acid sequences (v) to (vi)above are combined.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 22 include proteins which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 22, and which have substantially the same activity as the amino acidsequence represented by SEQ ID NO. 22.

Examples of substantially the same activity include receptor-bindingactivity, signal-transduction activity, the hypotensive activitydescribed in Example 1 below, and the smooth muscle contracting activitydescribed in Example 2 below. “Substantially the same” means that theactivity is the same in terms of characteristics. As such, thereceptor-binding activity, signal-transduction activity, or the likeshould be the same (for example, about 0.01 to 100-fold, preferablyabout 0.5 to 20-fold, and more preferably about 0.5 to 2-fold), althoughthe quantitative factors such as the extent of the activity or thepolypeptide molecular weight may vary.

Activity such as the receptor-binding activity or signal-transductionactivity can be assayed in accordance with methods that are well knownper se, such as the screening method described below.

Specific examples of polypeptides with an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 22 include the polypeptides described in“Detailed Description of Polypeptides of the Invention 4” and “DetailedDescription of Polypeptides of the Invention 5” below, but specificpreferred examples include:

(i) polypeptides consisting of an amino acid sequence represented by SEQID NO. 22;

(ii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 23;

(iii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 24;

(iv) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 25;

(v) polypeptides consisting of an amino acid sequence represented by SEQID NO. 26;

(vi) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 27;

(vii) polypeptides consisting of an amino acid sequence represented bySEQ ID NO. 28; and

(viii) polypeptides in which the C terminal carboxyl groups of thepolypeptides in (i) through (vii) above are amidated.

Detailed Description of Polypeptides of the Invention 4

More specific examples of “polypeptides with an amino acid sequence thatis the same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 22” include:

(6) polypeptides with an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 20.

The polypeptide with an amino acid sequence represented by SEQ ID NO. 20is ATT#21F.

“Polypeptides comprising an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 20” can comprise any other amino acid sequence, provided that theyalso comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 20.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 20 include amino acidsequences which comprise an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 22, and which have about at least 60%, more preferably about atleast 70%, even more preferably about at least 80%, and still morepreferably about at least 90% homology with the amino acid sequencerepresented by SEQ ID NO. 20.

Examples of amino acid sequences that are substantially the same as theamino acid sequence represented by SEQ ID NO. 20 include:

(i) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 22, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) of theamino acids in the amino acid sequence represented by SEQ ID NO. 20 havebeen deleted;

(ii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID No. 22, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids have been added to the amino acid sequence represented bySEQ ID NO. 20;

(iii) an amino acid sequence comprising an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 22, in which 1 or 2 or more (preferably 1 toabout 20, more preferably 1 to about 10, and even more preferably 1 to5) amino acids in the amino acid sequence represented by SEQ ID NO. 20have been substituted with other amino acids;

(iv) amino acid sequences in which amino acid sequences (i) through(iii) above are combined;

(v) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 22, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids (residues) other than those at positions 85 (Phe), 87 (Gly), 88(Leu), and 89 (Leu) from the N terminal in the amino acid sequencerepresented by SEQ ID NO. 20 have been deleted;

(vi) amino acid sequences comprising an amino acid sequence that is thesame as or substantially the same as the amino acid sequence representedby SEQ ID NO. 22, in which 1 or 2 or more (preferably 1 to about 20,more preferably 1 to about 10, and even more preferably 1 to 5) aminoacids other than those at positions 85 (Phe), 87 (Gly), 88 (Leu), and 89(Leu) from the N terminal in the amino acid sequence represented by SEQID NO. 20 have been substituted with other amino acids; and

(vii) amino acid sequences in which amino acid sequences (v) to (vi)above are combined.

Preferred examples of polypeptides with an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 20 include peptides which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 22, and which have substantially the same activity as the amino acidsequence represented by SEQ ID NO. 20.

Examples of substantially the same activity include receptor-bindingactivity and signal-transduction activity. “Substantially the same”means that the activity is the same in terms of characteristics.However, because polypeptides which have an amino acid sequence that issubstantially the same as the amino acid sequence represented by SEQ IDNO. 20 may also be obtained in the form of ATT, ATTshort1, as well asATTshort2 precursor polypeptides, they need not necessarily have thephysiological activity of ATT, ATTshort1, and ATTshort2 (such as thehypotensive activity described in Example 1 below or the smooth musclecontracting activity described in Example 2 below).

Specific preferred examples of polypeptides with an amino acid sequencethat is the same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 20 include polypeptides comprising an aminoacid sequence represented by SEQ ID NO. 20.

Detailed Description of Polypeptides of the Invention 5

Specific examples of “polypeptides with an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 17” and “polypeptides with an amino acidsequence that is the same as or substantially the same as the amino acidsequence represented by SEQ ID NO. 32”:

(7) polypeptides with an amino acid sequence that is the same as orsubstantially the same as the amino acid sequence represented by SEQ IDNO. 17 and an amino acid sequence that is the same as or substantiallythe same as the amino acid sequence represented by SEQ ID NO. 32.

“Amino acid sequences that are the same as or substantially the same asthe amino acid sequence represented by SEQ ID NO. 17,” “amino acidsequences that are the same as or substantially the same as the aminoacid sequence represented by SEQ ID NO. 32,” and polypeptides comprisingthose amino acid sequences are as described above.

Specific preferred examples of polypeptides comprising an amino acidsequence that is the same as or substantially the same as the amino acidsequence represented by SEQ ID NO. 17 and an amino acid sequence that isthe same as or substantially the same as the amino acid sequencerepresented by SEQ ID NO. 32 include polypeptides consisting of theamino acid sequence represented by SEQ ID NO. 20.

Method for Producing Polypeptides of the Invention

Polypeptides of the invention can be produced from the aforementionedhuman and mammal cells or tissue by polypeptide purification methodsthat are well known per se, and can be produced by culturingtransformants that have been transformed with polynucleotides (DNA)coding for polypeptides of the invention as described below. They canalso be produced by the following methods of protein (polypeptide)synthesis or methods based thereon.

When the polypeptides are produced from the cells or tissue of humans ormammals, the human or mammal cells or tissue can be homogenized and thenextracted with an acid or the like, and the extract can be purified andisolated by a combination of reverse phase chromatography, ion exchangechromatography, or the like.

Commercially available protein synthesis resins can be used in thesynthesis of polypeptide amides in the present invention (as notedabove, polypeptide amides of the invention are sometimes referred to forthe sake of convenience as “polypeptides of the invention”). Examples ofsuch resins include chloromethyl resin, hydroxymethyl resin,benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcoholresin, 4-methylbenzhydrylamine resin, PAM resin,4-hydroxymethylmethylphenylacetamidomethyl resin, polyacrylamide resin,4-(2′,4′-dimethoxyphenyl-hydroxymethyl)phenoxy resin, and4-(2′,4′-dimethoxyphenyl-Fmoc aminoethyl)phenoxy resin. The use of suchresins allows amino acids having α-amino groups and side chainfunctional groups protected by suitable protective groups to becondensed on the resin according to the sequence of the target peptideby any of various methods of condensation, which are well-known per se.After the reaction, the polypeptide can ultimately be cut out of theresin, the various protective groups can be simultaneously removed, anda reaction can be brought about to form intramolecular disulfide bondsin a highly diluted solution, giving the target polypeptides or amidesthereof.

Various activating reagents which can be used for peptide synthesis canbe employed in the condensation of the aforementioned protected aminoacids, although carbodiimides are particularly preferred. Examples ofsuch carbodiimides include DCC, N,N′-diisopropylcarbodiimide, andN-ethyl-N′-(3-dimethylaminopropyl)carbodiimide. To activate synthesiswith such a reagent, a racemization inhibitor additive (such as HOBt orHOOBt) and the protected amino acids can be added to the resin directly,or the inhibitor can be added to the resin after the activation of theprotected amino acids, in the form of a symmetric acid anhydride or anHOBt ester or HOOBt ester.

Solvents which can be used for the activation of protected amino acidsor their condensation with the resin may be selected from known solventswhich can be used in peptide condensation. Examples of such solventsinclude acid amides such as N,N-dimethyl formamide, N,N-dimethylacetamide, and N-methyl pyrrolidone, halohydrocarbons such as methylenechloride and chloroform, alcohols such as trifluoroethanol, sulfoxidessuch as dimethyl sulfoxide, ethers such as pyridine, dioxane, andtetrahydrofuran, nitriles such as acetonitrile and propionitrile, esterssuch as methyl acetate and ethyl acetate, or suitable mixtures thereof.The reaction temperature is selected from within the known range knownto allow the formation of protein bonds, which is usually between about−20° C. to 50° C. Activated amino acid derivatives are generally used ina proportion of 1.5- to 4-fold excess. When tests employing a commonninhydrin reaction reveal insufficient condensation, the condensationreaction can be repeated without removing the protective groups untilsufficient condensation has been achieved. When repeated condensationfails to result in sufficient condensation, the unreacted amino acidscan be acetylated using acetic anhydride or acetylimidazole.

Protective groups for the starting material amino groups include Z, Boc,tert-pentyloxycarbonyl, isobornyloxycarbonyl,4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl,trifluoroacetyl, phthaloyl, formyl, 2-nitrophenylsulfenyl,diphenylphosphinothioyl, and Fmoc.

Carboxyl groups can be protected, for example, by alkyl esterification(such as methyl, ethyl propyl, butyl, t-butyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, 2-adamantyl or similar linear, branched, orcyclic alkyl esterification), aralkyl esterification (such as benzylester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester,and benzhydryl esterification), and phenacyl esterification,benzyloxycarbonyl hydrazidation, tert-butoxycarbonyl hydrazidation, andtrityl hydrazidation.

Hydroxyl groups of serine can be protected, for example, throughesterification or etherification. Groups suitable for suchesterification include lower alkanoyl groups such as acetyl, aroylgroups such as benzoyl, and carbonic acid-derived groups such asbenzyloxycarbonyl and ethoxycarbonyl. Groups suitable for etherificationinclude benzyl, tetrahydropyranyl, and t-butyl.

Protective groups for the phenolic hydroxyl group of tyrosine includeBzl, Cl₂-Bzl, 2-nitrobenzyl, Br-Z, and t-butyl.

Examples of protective groups for the imidazole of histidine includeTos, 4-methoxy-2,3,6-trimethylbenzenesulfonyl, DNP, benzyloxymethyl,Bum, Boc, Trt, and Fmoc.

Examples of activated starting material carboxyl groups include thecorresponding acid anhydrides, azides, and activated esters (esters ofalcohols (such as pentachlorophenol, 2,4,5-trichlorophenol,2,4-dinitrophenol, cyanomethyl alcohol, para-nitrophenol, HONB,N-hydroxysuccimide, N-hydroxyphthalimide, and HOBt)). Examples ofactivated starting material amino groups include the correspondingphosphoric amides.

Methods for eliminating (removing) protective groups include catalyticreduction in a hydrogen current in the presence of a catalyst such as Pdblack or Pd-carbon, acid treatment with anhydrous hydrogen fluoride,methanesulfonic acid, trifluoromethanesulfonic acid, trifluoroaceticacid or mixtures thereof, treatment with a base such asdiisopropylethylamine, triethylamine, piperidine, or piperazine, orreduction with sodium in liquid ammonia. Elimination reactions by theaforementioned acid treatment are generally brought about at atemperature of about −20 to 40° C., but it is effective to add a cationscavenger such as anisole, phenol, thioanisole, meta-cresol,para-cresol, dimethylsulfide, 1,4-butanedithiol or 1,2-ethanedithiolduring the acid treatment. The 2,4-dinitrophenyl group used as aprotective group for the imidazole of histidine is eliminated bythiophenol treatment, and the formyl group used as the protective groupfor the indole of tryptophan may be deprotected by acid treatment in thepresence of the aforementioned 1,2-ethanedithiol, 1,4-butanedithiol, orthe like, and can also be removed by alkali treatment with dilute sodiumhydroxide solution, dilute ammonia, or the like.

The protection and deprotection of functional groups which cannot beinvolved in the reaction of the starting materials, the elimination ofthe protective groups, the activation of functional groups involved inthe reaction, and the like can be selected from the appropriate knowngroups and methods.

Another method for obtaining polypeptide amides is to first protect theα-carboxyl groups of the carboxyl terminal amino acids by amidation, andto then extend the peptide (protein) chain on the amino group side tothe desired length, producing proteins in which only the protectivegroups for the α-amino groups of the N terminal of the peptide chainhave been removed and polypeptides in which only the protective groupsof the carboxyl groups of the C terminal have been removed in order tocondense both proteins in the aforementioned solvent mixture. Thedetails of condensation are the same as above. The protectedpolypeptides obtained by condensation are purified, and all theprotective groups can be removed by the aforementioned methods to obtainthe desired crude polypeptides. The crude polypeptides can be purifiedby a number of well known techniques for that purpose, and the primaryfractions can be lyophilized to give the desired polypeptide amides.

An example of a way to obtain polypeptide esters (as noted above,polypeptide esters of the present invention are sometimes referred tofor the sake of convenience as “polypeptides of the invention” in thepresent Specification) is to condense the α-carboxyl groups of thecarboxyl terminal amino acids with a desired alcohol to form an aminoacid ester, and to then obtain the desired polypeptide ester in the samemanner as polypeptide amides.

Polypeptides of the present invention can be produced by methods ofpeptide synthesis that are well-known per se. The peptides can besynthesized in either the solid or liquid phase, for example. In otherwords, the target peptide can be produced upon the condensation of apartial polypeptide or amino acid capable of forming a polypeptide ofthe invention with the remainder, and the subsequent elimination of anyprotective groups when the product has protective groups. The methods in(1) through (5) below are examples of commonly known methods ofcondensation and methods for eliminating protective groups in suchcases.

(1) M. Bodanszky and M. A. Ondetti: Peptide Synthesis, IntersciencePublishers, New York (1966);

(2) Schroeder and Luebke: The Peptide, Academic Press, New York (1965);

(3) Nobuo Izumiya et al.: Fundamentals and Experiments in PeptideSynthesis, Maruzen (1975);

(4) Haru'aki Yajima and Shunpei Sakakibara: Biochemical ExperimentSeries 1: Protein Chemistry IV, 205 (1977); and

(5) Haru'aki Yajima (ed.), Development of Drugs-Continued, 14, PeptideSynthesis, Hirokawa Shoten.

Following the reaction, the partial peptides of the present inventioncan be purified and isolated by a combination of common methods ofpurification such as solvent extraction, distillation, columnchromatography, liquid chromatography, and recrystallization.Polypeptides obtained in free form by the above method can be convertedto a suitable salt by a common method. Conversely, polypeptides obtainedin the form of salts can be converted to free form by a common method.

Description of Polynucleotides of the Invention

Examples of polynucleotides coding for polypeptides of the inventioninclude any comprising a base sequence (DNA or RNA, and preferably DNA)coding for the aforementioned polypeptides of the invention. Examples ofsuch polynucleotides include DNA or RNA, such as mRNA, coding forpolypeptides of the present invention, and may be either double- orsingle-stranded. Double-stranded examples include double-stranded DNA,double-stranded RNA, or DNA:RNA hybrids. Single-stranded examplesinclude sense strands (that is, coding strands) and antisense strands(noncoding strands).

mRNA of polypeptides of the present invention can be assayed by a methodknown in the literature (“New PCR and Applications” 15(7) (1997)) or amethod based thereon using polynucleotides, or portions thereof, thatcode for polypeptides of the invention.

Examples of DNA coding for polypeptides of the invention include genomicDNA, genomic DNA libraries, cDNA of the aforementioned tissue and cells,cDNA libraries of such tissue and cells, and synthetic DNA. Vectors usedin libraries may be any from among bacteriophages, plasmids, cosmids,phagemids, and the like. Total RNA and mRNA fractions prepared from suchtissue and cells can also be amplified directly by reverse transcriptasepolymerase chain reaction (RT-PCR).

Examples of DNA coding for polypeptides of the present invention includeDNA with the base sequence represented by SEQ ID NO. 30 or 33, or DNAwhich has a base sequence hybridizable under highly stringent conditionswith the base sequence represented by SEQ ID NO. 30 or 33, and whichcodes for a receptor having substantially the same activity (such asreceptor-binding activity, signal-transduction activity, the hypotensiveactivity described in Example 1 below, or the smooth muscle contractingactivity described in Example 2 below) as polypeptides of the presentinvention.

Examples of DNA capable of hybridizing under highly stringent conditionswith the base sequence represented by SEQ ID NO. 30 or 33 include DNAcontaining a base sequence with at least about 70%, preferably at leastabout 80%, more preferably at least 90%, and even more preferably atleast 95% homology with the base sequence represented by SEQ ID NO. 30or 33.

Hybridization can be managed in accordance with methods that arewell-known per se or methods based thereon, such as the methods given inMolecular Cloning 2^(nd) Ed. (J. Sambrook et al., Cold Spring HarborLab. Press (1989)). When a commercially available library is used, themethod given in the accompanying protocol should be followed.Hybridization is more preferably capable of being managed under highlystringent conditions.

Highly stringent conditions refer to conditions involving, for example,a sodium concentration of about 19 to 40 mM, and preferably about 19 to20 mM, and a temperature of about 50 to 70° C., and preferably about 60to 65° C. A sodium concentration of about 19 mM and a temperature ofabout 65° C. are ideal.

More specific examples of polynucleotides (DNA) coding for polypeptidesof the invention include the following:

(1) DNA with a base sequence represented by SEQ ID NO. 30 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 17;

(2) DNA with a base sequence represented by SEQ ID NO. 33 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 32;

(3) DNA with a base sequence represented by SEQ ID NO. 29 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 7;

(4) DNA with a base sequence represented by SEQ ID NO. 4 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 3;

(5) DNA with a base sequence represented by SEQ ID NO. 14 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 13;

(6) DNA with a base sequence represented by SEQ ID NO. 21 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 20;

(7) DNA with a base sequence represented by SEQ ID NO. 31 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 22;

(8) DNA with a base sequence represented by SEQ ID NO. 40 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 23;

(9) DNA with a base sequence represented by SEQ ID NO. 41 may be used asDNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 24;

(10) DNA with a base sequence represented by SEQ ID NO. 42 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 25;

(11) DNA with a base sequence represented by SEQ ID NO. 43 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 26;

(12) DNA with a base sequence represented by SEQ ID NO. 44 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 27;

(13) DNA with a base sequence represented by SEQ ID NO. 45 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 28;

(14) DNA with a base sequence represented by SEQ ID NO. 46 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 34;

(15) DNA with a base sequence represented by SEQ ID NO. 47 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 35;

(16) DNA with a base sequence represented by SEQ ID NO. 48 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 36;

(17) DNA with a base sequence represented by SEQ ID NO. 49 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 37;

(18) DNA with a base sequence represented by SEQ ID NO. 50 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 38; and

(19) DNA with a base sequence represented by SEQ ID NO. 51 may be usedas DNA coding for polypeptides of the invention having an amino acidsequence represented by SEQ ID NO. 39.

Polynucleotides comprising a portion of the base sequence of DNA codingfor polypeptides of the invention or a portion of a base sequencecomplementary to such DNA is used in the sense including both DNA aswell as RNA coding for partial peptides of polypeptides in theinvention.

According to the present invention, antisense polynucleotides (nucleicacid) capable of inhibiting the replication or expression of polypeptidegenes of the invention can be designed and synthesized on the basis ofcloned or sequenced inventive polypeptide-encoding DNA base sequencedata. Such polynucleotides (nucleic acids) can hybridize with the RNA ofpolypeptide genes of the invention and can inhibit RNA synthesis orfunction or can interact with polypeptide-related RNA of the inventionto regulate and inhibit the expression of polypeptide gene in theinvention. Polynucleotides complementary to the selected base sequenceof polypeptide-associated RNA in the invention or polynucleotidescapable of specifically hybridizing with polypeptide-associated RNA inthe invention are useful in regulating and inhibiting the in vivo and invitro expression of polypeptide genes of the invention, which can beuseful for the treatment or diagnosis of various diseases. The term“corresponding” means complementary to or having homology with aspecific sequence of nucleic acids, base sequences, or nucleotides,including genes. “To correspond” between nucleotides, base sequences, ornucleic acid and peptides (proteins) generally indicates amino acids ofthe target peptide (proteins) induced from the nucleotide (nucleic acid)sequence or complement. The inventive polypeptide gene 5′ end hairpinloop, 5′ end 6-base-pair repeats, 5′ end untranslated region,polypeptide translation initiation codon, protein coding region, ORFtranslation initiation codon, 3′ untranslated region, 3′ end palindromeregion, and 3′ end hairpin loop can be selected as preferred targetregions, although any region within the polypeptide genes of theinvention can be selected as targets.

“Antisense” refers to the relationship between the target nucleic acidand polynucleotides that are complementary to at least a portion of thetarget region, that is, the relationship between targets andhybridizable polynucleotides. Examples of antisense polynucleotidesinclude polydeoxynucleotides containing 2-deoxy-D-ribose,polydeoxynucleotides containing D-ribose, and other types ofpolynucleotides comprising N-glycosides of purine or pyrimidine bases,or other polymers with non-nucleotide skeletons (such as commerciallyavailable protein nucleic acids and synthetic sequence-specific nucleicacid polymers) or other polymers with special linkages (provided thatthe polymers contain nucleotides with configurations permitting the kindof base pairing or base stacking found in DNA and RNA). They may includedouble- and single-stranded DNA, double- and single-stranded RNA, andDNA:RNA hybrids, as well as unmodified polynucleotides (or unmodifiedoligonucleotides) and those with known modifications, such as those withlabels which are known in the art, those that are capped, those thathave been methylated, those in which one or more natural nucleotideshave been substituted with analogues, those with intramolecularnucleotide modifications such as those with uncharged linkages (forexample, methyl phosphonates, phosphotriesters, phosphoramidates, andcarbamates) and those with charged linkages or sulfur-containinglinkages (for example, phosphorothioates and phosphorodithioates), thosewith pendant moieties, such as proteins (including nucleases, nucleaseinhibitors, toxins, antibodies, signal peptides, and poly-L-lysine) andsaccharides (such as monosaccharides), those with intercalators (such asacridine and psoralen), those containing chelators (such as metals,radioactive metals, boron, and oxidative metals), those containingalkylators, and those with modified linkages (such as a anomeric nucleicacids). “Nucleosides,” “nucleotides,” and “nucleic acids” include thosewith purine and pyrimidine bases as well as other modified heterocyclicbases. Such modifications include methylated purines and pyrimidines,acylated purines and pyrimidines, and other heterocycles. The sugarmoieties of modified nucleosides or nucleotides may also be modified,such as the substitution of one or more hydroxyl with halogens,aliphatic groups, or the like, or their conversion to functional groupssuch as ethers or amines.

The antisense polynucleotides (nucleic acids) of the present inventioncomprise RNA, DNA, or modified nucleic acids (RNA, DNA). Specificexamples of modified nucleic acids include, but are not limited to,nucleic acid sulfur derivatives or thiophosphate derivatives andpolynucleoside amides or oligonucleoside amides resistant todegradation. The antisense nucleic acids in the present invention shouldbe designed to achieve the following. Specifically, the antisensenucleic acids should have greater intracellular stability, the antisensenucleic acids should have higher cell permeability, they should havegreater affinity for the target sense strand, and the toxicity of anytoxic antisense nucleic acids should be minimized.

Many such modifications are known in the art. Examples can be found inJ. Kawakami et al., Pharm Tech Japan, Vol. 8, pp. 247 (1992) and Vol. 8,pp. 395 (1992), and S. T. Crooke et al. ed., Antisense Research andApplications, CRC Press (1993).

The antisense nucleic acids of the invention can contain altered ormodified sugars, bases, or linkages, they can be delivered in specialsystems such as liposomes and microspheres, they can be employed in genetherapy, or they can be given in conjugated form. Examples of those usedin conjugated form include those conjugated to polycations such aspolylysine which serve to neutralize the charge of phosphate skeletons,and substances (such as phospholipids and cholesterol) which enhanceinteraction with cell membranes or bring about greater nucleic aciduptake. Preferred lipids for conjugation include cholesterol orderivatives thereof (such as cholesteryl chloroformate and cholic acid).These can be conjugated to the 3′ or 5′ ends of nucleic acids, and canalso be conjugated through bases, sugars, or intramolecular nucleosidelinkages. Examples of other groups include capping groups specificallyconfigured to the 3′ or 5′ ends of nucleic acids, preventing degradationby nucleases such as exonuclease or RNase. Such capping groups include,but are not limited to, hydroxyl protecting groups known in the art,including glycols such as polyethylene glycol and tetraethylene glycol.

The inhibiting activity of antisense nucleic acids can be studied usingtransformants of the invention, in vivo or in vitro gene expressionsystems of the invention, and in vivo or in vitro translation systems ofpolypeptides in the invention. Such nucleic acids can be applied tocells by a variety of methods that are well known per se.

The aforementioned antisense nucleic acids include those that inhibitthe synthesis or function of DNA or RNA of the polypeptide genes in theinvention upon hybridization with such DNA or RNA as well as uponhybridization with the untranslated regions (such as polynucleotidesrepresented by the base sequence from positions 1 through 135 orpositions 364 through 720 from the 5′ end of the base sequencerepresented by SEQ ID NO. 5) of polypeptides of the invention.

DNA Cloning

Procedures for cloning DNA coding for polypeptides of the presentinvention include using synthetic DNA primers containing a portion of abase sequence of a polypeptide in the invention for amplification byPCR, or screening DNA incorporated in suitable vectors by hybridizationwith labeled synthetic DNA or DNA fragments coding for some or allregions of polypeptides of the present invention. Hybridization shouldbe managed in accordance with the methods given, for example, inMolecular Cloning 2^(nd) Ed. (J. Sambrook et al., Cold Spring HarborLab. Press (1989)). When a commercially available library is used, themethod given in the accompanying protocol should be followed.

The DNA base sequence can be modified by methods that are known per seor methods based on them, such as ODA-LA PCR, the gapped duplex method,or the Kunkel method, using a common kit such as Mutan™-Super Express Km(Takara Shuzo) or Mutan™-K (Takara Shuzo).

The cloned DNA coding for a polypeptide of the invention can be used assuch or after being digested with the desired restriction enzymes orafter the addition of linker DNA, depending on the intended purpose. TheDNA may have ATG as the translation initiation codon on the 5′ terminalside, and TAA, TGA, or TAG as the translation termination codon on the3′ terminal side. The translation initiation and termination codons canbe added using suitable synthetic DNA adapters.

Expression vectors for polypeptides in the present invention can beprepared, for example, by (a) cutting out the target DNA fragment fromDNA coding for a polypeptide of the present invention, and (b) ligatingthe DNA fragment downstream of a promoter in a suitable expressionvector.

Examples of vectors which can be used include E. coli plasmids (such aspBR322, pBR325, pUC12, and pUC13), Bacillus subtilis plasmids (such aspUB110, pTP5, and pC194), yeast plasmids (such as pSH19 and pSH15),bacteriophages such as λ phages, and animal viruses such asretroviruses, vaccinia viruses, and baculoviruses, as well as pA1-11,pXT1, pRc/CMV, pRc/RSV, and pcDNA I/Neo.

Promoters that can be used in the present invention include any that aresuitable for the host which is used to express the gene. Examples foranimal cell hosts include SRα promoters, SV40 promoters, LTR promoters,CMV promoters, and HSV-TK promoters.

CMV promoters and SRα promoters are preferred among these. Promotersthat are preferred for E. coli hosts include trp promoters, lacpromoters, recA promoters, λP_(L) promoters, and lpp promoters; examplesthat are preferred for Bacillus hosts include SPO1 promoters, SPO2promoters, and penp promoters; and examples that are preferred for yeasthosts include PHO5 promoters, PGK promoters, GAP promoters, and ADHpromoters. Examples that are preferred for insect cell hosts includepolyhedrin and P10 promoters.

In addition to the above, expression vectors can also contain enhancers,splicing signals, poly A linker signals, selection markers, SV40replication origins (sometimes referred to below as SV40ori), and thelike as needed. Examples of selection markers include the dihydrofolatereductase (sometimes referred to below simply as dhfr) gene{methotrexate (MTX) resistance}, ampicillin resistance gene (sometimesreferred to below simply as Amp^(r)), and neomycin resistance gene (G418resistance, sometimes referred to below simply as Neo^(r)). Inparticular, the target gene can also be selected with thymidine-freemedium when the dhfr gene is used as the selection marker withCHO(dhfr⁻) cells.

A signal sequence compatible with the host can be attached to theN-terminal side of polypeptides in the present invention if needed.Examples of signal sequences for Escherichia hosts include PhoA signalsequences and OmpA signal sequences; examples for Bacillus hosts includeα-amylase signal sequences and subtilisin signal sequences; examples foryeast hosts include MFα signal sequences and SUC2 signal sequences; andexamples for animal cell hosts include insulin signal sequences,α-interferon signal sequences, and antibody molecule signal sequences.

The resulting vectors containing DNA coding for polypeptides of thepresent invention can be used to produce transformants.

Examples of hosts that can be used include Escherichia microorganisms,Bacillus microorganisms, yeasts, insect cells, insects, and animalcells.

Specific examples of Escherichia microorganisms include Escherichia coliK12-DH1 (Proc. Natl. Acad. Sci. USA, Vol. 60, 160 (1968)), JM103(Nucleic Acids Research, Vol. 9, 309 (1981)), JA221 (Journal ofMolecular Biology, Vol. 120, 517 (1978)), HB101 (Journal of MolecularBiology, Vol. 41, 459 (1969)), and C600 (Genetics, Vol. 39, 440 (1954)).

Examples of Bacillus microorganisms include Bacillus subtilis MI114(Gene, Vol. 24, 255 (1983)) and 207-21 (Journal of Biochemistry, Vol.95, 87 (1984)).

Examples of yeasts include Saccharomyces cerevisiae AH22, AH22R⁻,NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913 andNCYC2036, and Pichia pastoris.

Examples of insect cells for the AcNPV virus include established celllines from the larvae of Spodoptera frugiperda (Sf cells), MG1 cellsfrom the interior lining of the gut of Trichoplusia ni, High Five™ cellsfrom Trichoplusia ni ova, Mamestra brassicae cells, and Estigmena acreacells. Examples for the BmNPV virus include the Bombyx mori cell line(Bombyx mori N; BmN cells). Examples of Sf cells include Sf9 cells (ATCCCRL 1711) and Sf21 cells (both by J. L. Vaughn et al., In Vivo, 13,213-217(1977)).

Examples of insects include silkworm larvae (Maeda et al, Nature, Vol.315, 592 (1985)).

Examples of animal cells include monkey cells COS-7, Vero, Chinesehamster ovary cells (CHO cells), dhfr gene-deficient Chinese hamsterovary cells (CHO (dhfr⁻) cells) mouse L cells, mouse AtT-20, mousemyeloma cells, rat GH3, and human FL cells.

Escherichia microorganisms can be transformed in accordance with methodsas disclosed in, for example, Proc. Natl. Acad. Sci. USA, Vol. 69, 2110(1972), and Gene, Vol. 17, 107 (1982). Bacillus microorganisms can betransformed in accordance with methods as disclosed in, for example,Molecular & General Genetics, Vol. 168, 111 (1979).

Yeasts can be transformed in accordance with methods as disclosed in,for example, Methods in Enzymology, Vol. 194, 182-187 (1991), and Proc.Natl. Acad. Sci. USA, Vol. 75, 1929 (1978).

Insect cells or insects can be transformed in accordance with methods asdisclosed in, for example, Bio/Technology, Vol. 6, 47-55 (1988).

Animal cells can be transformed by methods as disclosed in, for example,Saibo Kogaku [Cell Engineering] Special Edition No. 8: Shin Saibo KogakuJikken Purotokoru [New Cell Engineering Experimental Protocols],263-267, published by Shujunsha (1995), and Virology, Vol. 52, 456(1973).

In this manner it is possible to obtain transformants which have beentransformed in expression vectors that contain DNA coding forpolypeptides of the invention.

Liquid media are preferred for the culture of transformants obtainedwith Escherichia or Bacillus hosts, and should be prepared with thecarbon sources, nitrogen sources, minerals, and the like which areneeded for the growth of the transformants. Examples of carbon sourcesinclude glucose, dextrin, soluble starch, and sucrose. Examples ofnitrogen sources include organic or inorganic substances such asammonium salts, nitrates, corn steep liquor, peptone, casein, meatextract, soybean cake, and potato extract. Examples of minerals includecalcium chloride, sodium dihydrogen phosphate, and magnesium chloride.Yeast extract, vitamins, growth-promoting factors, and the like may alsobe added. The medium pH is preferably about 5 to 8.

The preferred medium for culturing Escherichia microorganisms is M9medium containing, for example, glucose and casamino acid (Miller,Journal of Experiments in Molecular Genetics, pp. 431-433, Cold SpringHarbor Laboratory, New York (1972)). The medium may be supplemented asneeded with drugs such as 3β-indolyl acrylic acid in order to ensurepromoter efficiency. Escherichia hosts can usually be cultured for about3 to 24 hours at about 15 to 43° C., while stirred and aerated asneeded.

Bacillus hosts can usually be cultured for about 6 to 24 hours at about30 to 40° C., while aerated or stirred as needed.

Examples of media for the culture of transformants using yeast hostsinclude Burkholder minimum medium (K. L. Bostian et al., Proc. Natl.Acad. Sci. USA, Vol. 77, 4505 (1980)), and SD medium containing 0.5%casamino acid (G. A. Bitter et al., Proc. Natl. Acad. Sci. USA, Vol. 81,5330 (1984)). The medium pH should be adjusted to between about 5 and 8.Culture usually lasts about 24 to 72 hours at about 20 to 35° C., whileaerated and stirred as needed.

Examples of media for the culture of transformants using insect cellhosts or insect hosts include Grace's insect medium (T. C. C. Grace,Nature, 195, 788 (1962)) suitably supplemented with an additive such as10% inactivated bovine serum. The medium pH should be adjusted tobetween about 6.2 and 6.4. Culture usually lasts about 3 to 5 days atabout 27° C., while aerated and stirred as needed.

Examples of media for the culture of transformants using animal cellhosts include MEM medium supplemented with about 5 to 20% fetal calfserum (Science, Vol. 122, 501 (1952)), DMEM medium (Virology, Vol. 8,396 (1959)), RPMI 1640 medium (Journal of the American MedicalAssociation, Vol. 199, 519 (1967)), and 199 medium (Proceedings of theSociety for the Biological Medicines, Vol. 73, 1 (1950)). The pH shouldbe between about 6 and 8. Culture usually lasts about 15 to 60 hours atabout 30 to 40° C., while aerated and stirred as needed.

In this manner it is possible to produce polypeptides of the presentinvention inside or outside the cells or cell membranes oftransformants.

The cloning and selection of cells obtained upon the chromosomalincorporation of expression vectors introduced to the aforementionedanimal cells is one way of ensuring the stable expression ofpolypeptides of the invention using animal cells. Specifically,transformants are selected using the aforementioned selection markers asindicators. Repeated cloning and selection of animal cells thus obtainedusing selection markers can result in stable animal cell lines with ahigh capacity for expressing polypeptides of the invention. When thedhfr gene is used as the selection marker, the culture can be managed asthe MTX concentration is gradually increased to select for resistance,allowing animal cell lines of even higher expression to be obtained uponthe intracellular amplification of DNA coding for polypeptides of theinvention together with the dhfr gene.

Polypeptides of the present invention can be isolated and purified fromthe above cultures in the following manner.

When polypeptides of the invention are extracted from cultured bacterialcells or cultured cells, the bacterial cells or cells are collected inthe usual manner after the culture and are suspended in a suitablebuffer, the cells are then disrupted by common ultrasonic treatment,lysozyme treatment, and/or freeze-thawing or the like, and a crudeextract of the polypeptides is then obtained by common centrifugation,filtration, or the like. The buffer may be supplemented with proteindenaturants such as urea or guanidine hydrochloride, or surfactant suchas Triton X-100™. When polypeptides of the invention are secreted in theculture, the cultured bacterial cells or cultured cells are separated inthe usual manner from the supernatant, and the supernatant is collected.

The polypeptides of the invention contained in the resulting culturesupernatant or extract can be purified by a suitable combination ofisolation and purification methods that are well-known per se. Examplesof such methods include: methods exploiting solubility, such as saltingout or solvent precipitation; methods primarily exploiting differencesin molecular weight, such as dialysis, ultrafiltration, gel filtrationand SDS-polyacrylamide gel electrophoresis; methods exploitingdifferences in electrical charge, such as ion-exchange chromatography;methods exploiting specific affinity, such as affinity chromatography;methods exploiting differences in hydrophobicity, such as reverse-phasehigh-performance liquid chromatography; and methods exploitingdifferences in isoelectric point, such as isoelectric focusing.

Polypeptides of the invention obtained in free form can be converted toa salt by a method that is well-known per se, or a method based thereon.Conversely, polypeptides obtained in the form of salts can be convertedto free form or to another salt by a method that is well-known per se,or a method based thereon.

Either before or after the purification of a polypeptide produced withrecombinants, a suitable protein-modifying enzyme can be allowed to actthereon in the usual manner to add any modifications or to removeportions of the polypeptide. Examples of such protein-modifying enzymesinclude trypsin, chymotrypsin, arginyl endopeptidase, protein kinase,and glycosidase. In polypeptides of the invention produced byrecombinants, the C terminal methionine may or may not be amidated,depending on the cells that are used. Those that are not amidated can beamidated by methods which are well known in the art.

The activity of the resulting polypeptides or their salts in theinvention can be assayed enzyme immunoassay or the like using specificantibodies and binding assays with labeled receptors.

Antibodies Against Polypeptides of the Invention

Antibodies against polypeptides in the invention can be any ofmonoclonal or polyclonal antibodies, provided that they can recognizepolypeptides of the invention.

Antibodies against polypeptides of the invention can be produced byusing polypeptides of the invention as antigen according to methods thatare well-known per se for producing antibodies and antiserum.

[Preparation of Monoclonal Antibodies]

(a) Preparation of Monoclonal Antibody-Producing Cells

Polypeptides of the present invention are administered, either alone oralong with a carrier and diluent, to mammals at a site permitting theproduction of antibodies. Freund's complete adjuvant or incompleteadjuvant may also be given in order to potentiate the production ofantibodies during administration. Administration is usually once every 2to 6 weeks, for a total of about 2 to 10 times. Mammals that can be usedinclude monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, andgoats, although the use of mice and rats is preferred.

In the preparation of cells which produce monoclonal antibodies,individual animals with suitable antibody titer can be selected from thewarm-blooded animals such as mice which have been immunized withantigen, the spleens or lymph nodes can be harvested 2 to 5 days afterfinal immunization, and the antibody-producing cells obtained therefromcan be fused with myeloma cells so as to prepare monoclonalantibody-producing hybridomas. The antibody titer in antiserum can beassayed, for example, by conducting a reaction between antiserum andlabeled polypeptides as described below, and by then assaying theactivity of the label binding to the antibody. Fusion can be managed,for example, in accordance with the method of Koehler and Milstein(Nature, Vol. 256, 495, (1975)). Examples of fusion promoters includepolyethylene glycol (PEG) and the Sendai virus, although the use of PEGis preferred.

Examples of myeloma cells include NS-1, P3U1, and SP2/0, although theuse of P3U1 is preferred. The proportion between the number ofantibody-producing cells (spleen cells) and the number of myeloma cellsis preferably about 1:1 to 20:1. Efficient cell fusion can be achievedby 1 to 10 minutes of incubation at about 20 to 40° C., and preferablyabout 30 to 37° C., with the addition of PEG (preferably, PEG 1000 toPEG 6000) in a concentration of about 10 to 80%.

A variety of methods can be employed to screen monoclonalantibody-producing hybridomas, such as methods in which hybridomaculture supernatant is added to a solid phase (such as a microplate) towhich the polypeptide antigen is adsorbed, either directly or with acarrier, and protein A or anti-immunoglobulin antibody (anti-mouseimmunoglobulin antibody when the cells used for cell fusion are from amouse) labeled with a radioactive substance, an enzyme, or the like isadded to detect monoclonal antibodies binding to the solid phase; ormethods in which hybridoma supernatant liquid is added to a solid phaseto which anti-immunoglobulin or protein A is adsorbed, and polypeptideslabeled with a radioactive substance, an enzyme, or the like are addedto detect monoclonal antibodies binding to the solid phase.

Monoclonal antibodies can be selected according to methods that arewell-known per se or methods based thereon. This can usually be done inanimal cell media containing HAT (hypoxanthine, aminopterin, andthymidine). Any medium allowing hybridomas to grow can be used forselection and growth. Examples include RPMI 1640 medium containing 1 to20%, and preferably 10 to 20%, fetal calf serum; GIT medium (Wako PureChemicals) containing 1 to 10% fetal calf serum; and serum-free medium(SFM-101, by Nissui Seiyaku) for hybridoma culture. The culturetemperature is usually 20 to 40° C., and preferably about 37° C. Theculture usually lasts from 5 days to 3 weeks, and preferably 1 to 2weeks. The culture can usually take place with 5% carbon dioxide gas.The antibody titer of the hybridoma culture supernatant can be assayedin the same manner as in the aforementioned assay of the antibody titerin antiserum.

(b) Purification of Monoclonal Antibodies

The monoclonal antibodies can be isolated and purified by common methodsfor isolating and purifying immunoglobulin in the same manner as theisolation and purification of polyclonal antibodies (such assalting-out, precipitation with alcohol, isoelectric precipitation,electrophoresis, adsorption and desorption using ion exchangers (such asDEAE), ultracentrifugation, gel filtration, and specific methods ofpurification in which only antibodies are collected using an activeadsorbent such as protein A or protein G, or an antigen-binding solidphase, and antibodies are obtained upon the dissociation of the bonds).

[Preparation of Polyclonal Antibodies]

Polyclonal antibodies of the present invention (meaning polyclonalantibodies against polypeptides of the invention) can be produced bymethods that are well-known per se or methods based thereon. Forexample, an immunogen (polypeptide antigen of the invention) and carrierprotein conjugate can be prepared, mammals can be immunized in the samemanner as in the production of monoclonal antibodies, substancescontaining antibody against polypeptides of the invention can beharvested from the immunized animals, and the antibodies can be isolatedand purified.

Any type of carrier protein can be crosslinked in any proportion in theimmunogen-carrier protein conjugate used to immunize mammals, as long asthey result in the efficient production of antibodies against haptenwhen crosslinked with the carrier for immunization. For example, bovineserum albumin, bovine thyroglobulin, or keyhole limpet hemocyanin can becoupled in a weight ratio of about 0.1 to 20, and preferably about 1 toabout 5, per unit hapten.

Various condensation agents can be used to couple the hapten andcarrier. Glutaraldehyde, carbodiimides, maleimide active esters, andactive ester reagents with thiol and dithiopyridyl groups can be used.

The condensation reaction product is administered, either alone or alongwith a carrier and diluent, to warm-blooded animals at a site permittingthe production of antibodies. Freund's complete adjuvant or incompleteadjuvant may also be given in order to potentiate the production ofantibodies during administration. Administration is usually once every 2to 6 weeks, for a total of about 3 to 10 times.

Polyclonal antibodies can be harvested from the blood, ascites fluid, orthe like of mammals immunized in the manner described above, and arepreferably harvested from the blood.

The antibody titer of the polyclonal antibodies in antiserum can beassayed in the same manner as in the assay of the antibody titer inserum described above. The polyclonal antibodies can be isolated andpurified in accordance with methods for the isolation and purificationof immunoglobulin in the same manner as the isolation and purificationof the aforementioned monoclonal antibodies.

Applications of Polypeptides and the Like of the Invention

The following are applications of (i) polypeptides of the invention,(ii) polynucleotides (DNA) coding for polypeptides of the invention(sometimes referred to below simply as polynucleotide (DNA)), (iii)antibodies against polypeptides of the invention (sometimes referred tobelow as antibodies of the invention), and (iv) antisense DNA.

(1) Treatment and/or Prevention of Diseases in which Polypeptides of theInvention are Involved

Polypeptides of the invention have physiological activity such ashypotensive activity or smooth muscle contracting activity, as noted inExamples 1 and 2 below.

Accordingly, it is highly possible that abnormal or missing DNA codingfor polypeptides of the present invention or abnormal or missing DNAcoding for receptor proteins of polypeptides in the invention are highlycould result in the onset of various diseases such as blood pressuredisorders (hypertension, for example), exocrine disorders, andcardiovascular disorders.

Polypeptides of the invention and polynucleotides (DNA) of the inventioncan therefore be used as drugs for the treatment and/or prevention ofsuch diseases, for example.

For example, in patients who lack or suffer from low levels of apolypeptide of the invention, the polypeptide and DNA of the inventioncan be returned to normal levels by (a) administering DNA of theinvention to the patient to bring about the in vivo expression of thepolypeptide of the invention, (b) inserting DNA of the invention intocells to bring about expression of the polypeptide of the invention, andthe cells can then be implanted in the patient, or (c) administering thepolypeptide of the invention to the patient to ensure that thepolypeptide functions satisfactorily or normally in the patient.

When DNA of the invention is used for the aforementioned therapeutic orprophylactic purposes, the DNA can be administered in the usual mannerto humans or warm-blooded animals, either on its own or after beinginserted in a suitable vector such as a retrovirus vector, adenovirusvector, or adenovirus-associated virus vector. The DNA of the inventioncan be given, either as such or in the form of a preparation with aphysiologically acceptable carrier such as an adjuvant to enhanceingestion, using a catheter such as a hydrogel catheter or gene gun.

When polypeptides of the present invention are used for the therapeuticand prophylactic purposes described above, they should be purified to atleast 90%, preferably to at least 95%, more preferably to at least 98%,and even more preferably to at least 99%.

The polypeptides of the present invention can be used, for example,orally in the form of optionally sugar-coated tablets, capsules,elixirs, microcapsules or the like, or they can be used parenterally inthe form of injections such as sterile solutions or suspensions withwater or other pharmaceutically acceptable liquids. These preparationscan be produced, for example, by mixing a polypeptide of the inventionwith physiologically acceptable carriers, flavoring agents, excipients,vehicles, antiseptics, stabilizers, binders, or the like, in the unitdose forms required in generally accepted pharmaceutical practice. Thecontent of the active ingredient in these preparations should give theappropriate dose within the specified range.

Examples of additives which can be mixed with tablets, capsules, and thelike include binders such as gelatin, corn starch, tragacanth, and gumarabic; excipients such as crystalline cellulose; extenders such as cornstarch, gelatin, and alginic acid; lubricants such as magnesiumstearate; sweetening agents such as sucrose, lactose, and saccharin; andflavoring agents such as peppermint, akamono oil, and cherry. In thecase of capsule unit dose forms, the aforementioned types of materialscan also include liquid carriers such as oils and fats. Sterilecompositions for injection can be formulated according to ordinarypharmaceutical practice such as the dissolution or suspension of activeingredients and naturally occurring vegetable oils such as sesame oil orcoconut oil in a vehicle such as water for injection.

Aqueous liquids for injection include physiological saline and isotonicsolutions containing glucose or other adjuvants (such as D-sorbitol,D-mannitol, and sodium chloride), and may be used in combination withappropriate dissolution aids such as alcohols (such as ethanol),polyalcohols (such as propylene glycol and polyethylene glycol), andnonionic surfactants (such as Polysorbate 80™ and HCO-50). Oleaginousliquids include sesame oil and soybean oil, and may be used incombination with benzyl benzoate, benzyl alcohol, or the like asdissolution aids. The above may also be blended with buffers (such asphosphate buffer and sodium acetate buffer), soothing agents (such asbenzalkonium chloride and procaine hydrochloride), stabilizers (such ashuman serum albumin and polyethylene glycol), preservatives (such asbenzyl alcohol and phenol), antioxidants, and the like. Suitable ampulesare usually aseptically filled with the resulting injection liquid.

Vectors having DNA of the invention may also be similarly prepared, andare usually used parenterally.

Because such preparations are safe and have low toxicity, they can beadministered, for example, to humans and warm-blooded animals (such asrats, mice, guinea pigs, rabbits, birds, sheep, pigs, cows, horses,cats, dogs, and monkeys).

The dosage of polypeptides of the present invention will vary dependingon the target disease, purpose of administration, route ofadministration or the like, but the daily adult oral dosage ofpolypeptides of the invention for the treatment of hypertension, forexample, may generally range from about 0.1 to 100 mg, preferably fromabout 1.0 to 50 mg, and even more preferably from about 1.0 to 20 mg, interms of polypeptides (per 60 kg body weight). The single parenteraldose of polypeptides will vary depending on the purpose ofadministration, target disease, and the like, but in the form of aninjection for adults, for example, the daily dosage of polypeptides ofthe invention for treatment of hypertension may usually range from about0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and even morepreferably about 0.1 to about 10 mg at a time, in terms of thepolypeptide of the present invention (per 60 kg body weight), given byinjection to the affected site. Doses for animals can be given ascalculated per 60 kg body weight.

(2) Screening of Candidate Drug Compounds for Diseases

As shown in Examples 1 and 2 below, the polypeptides of the presentinvention have physiological activity such as hypotensive activity andsmooth muscle contracting activity, and compounds or their salts in theinvention promoting these functions (specifically, agonists ofpolypeptides in the invention) can thus be used as drugs for thetreatment and/or prevention of diseases such as blood pressure disorders(such as hypertensions), exocrine disorders, and cardiovasculardiseases.

Compounds or their salts inhibiting such functions in the invention(specifically, antagonists of polypeptides in the invention) can beused, for example, as drugs for the treatment and/or prevention ofallergic diseases, asthma, angina pectoris, atherosclerosis, diabetes,hyperlipemia, emesis, bone diseases, pollakiuria, AIDS, and bloodpressure disorders.

Polypeptides of the invention can be used or recombinant polypeptideexpression systems of the present invention can be constructed for usein receptor or binding assay systems to screen compounds or their saltswhich modify binding between polypeptides of the present invention andtheir receptors (compounds that promote or inhibit the activity of thepolypeptides in the present invention) (such as peptides, proteins,non-peptide compounds, synthetic compounds, and fermented products) asagonists and antagonists. Such compounds or their salts includecompounds or their salts with polypeptide receptor-mediatedcell-stimulating activity (such as activity in promoting arachidonicacid release, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, changes in cell membrane potential, intracellularprotein phosphorylation, c-fos activation, and decreases in pH) (thatis, agonists of polypeptides in the present invention), and compounds ortheir salts with no such cell-stimulating activity (that is, antagonistsof polypeptides in the present invention). To “modify binding withreceptors” means to either inhibit or promote receptor binding.

That is, the present invention is intended to provide:

a method for screening compounds, or their salts, that promote orinhibit the activity of polypeptides of the invention, characterized bythe use of a polypeptide of the invention, specifically,

a method for screening compounds (compounds that promote or inhibit theactivity of polypeptides in the invention: agonists or antagonists ofpolypeptides in the invention) or their salts which modify bindingbetween polypeptides of the present invention and receptors ofpolypeptides of the invention, characterized by comparing (i) cases inwhich a polypeptide of the present invention is brought into contactwith a polypeptide receptor or salt thereof in the invention, or apartial peptide or its salt of a polypeptide receptor in the presentinvention, and (ii) cases in which a polypeptide of the presentinvention and a test compound are brought into contact with apolypeptide receptor or salt thereof in the invention, or a partialpeptide or its salt of a polypeptide receptor in the present invention.

The screening method of the present invention entails, for example,comparing assays of cell-stimulating activity or the extent to whichpolypeptides of the invention bind to polypeptide receptors or saltsthereof in the invention, or partial peptides or their salts ofpolypeptide receptors in the present invention between (i) cases inwhich a polypeptide of the present invention is brought into contactwith a polypeptide receptor or salt thereof in the invention, or apartial peptide or its salt of a polypeptide receptor in the presentinvention, and (ii) cases in which a polypeptide of the presentinvention and a test compound are brought into contact with apolypeptide receptor or salt thereof in the invention, or a partialpeptide or its salt of a polypeptide receptor in the present invention.

Examples of receptors for polypeptides of the present invention includethose among a variety of receptors, which have binding activity withpolypeptides of the present invention, and through which thepolypeptides of the present invention are found to have cell-stimulatingactivity on cells expressing such receptors (such as activity inpromoting arachidonic acid release, acetylcholine release, intracellularCa²⁺ release, intracellular cAMP production, intracellular cGMPproduction, inositol phosphate production, changes in cell membranepotential, intracellular protein phosphorylation, c-fos activation, anddecreases in pH).

Specific examples include:

(i) NK-1;

(ii) NK-2;

(iii) NK-3; and

(iv) NK-4.

Specific examples of such a screening method include:

(i) methods for screening compounds or their salts which modify bindingbetween polypeptides of the present invention and polypeptide receptorsof the present invention (compounds that promote or inhibit the activityof polypeptides of the present invention: agonists or antagonists ofpolypeptides in the invention), characterized by comparative assay ofthe extent to which labeled polypeptides of the present invention bindto polypeptide receptors or their salts, or partial peptides or theirsalts, between cases in which the labeled polypeptides of the presentinvention are brought into contact with polypeptide receptors or saltsthereof in the invention, or partial peptides or their salts ofpolypeptide receptors in the present invention, and cases in whichlabeled polypeptides of the present invention and test compounds arebrought into contact with polypeptide receptors or salts thereof in theinvention, or partial peptides or their salts of polypeptide receptorsin the present invention;

(ii) methods for screening compounds or their salts which modify bindingbetween polypeptides of the present invention and polypeptide receptorsof the present invention (compounds that promote or inhibit the activityof polypeptides of the present invention: agonists or antagonists ofpolypeptides in the invention), characterized by comparative assay ofthe extent to which labeled polypeptides of the present invention bindto cells or membrane fractions between cases in which labeledpolypeptides of the present invention are brought into contact withcells or cell membrane fractions containing the polypeptide receptors ofthe present invention, and cases in which labeled polypeptides of thepresent invention and test compounds are brought into contact with cellsor cell membrane fractions containing the polypeptide receptors of thepresent invention;

(iii) methods for screening compounds or their salts which modifybinding between polypeptides of the present invention and polypeptidereceptors of the present invention (compounds that promote or inhibitthe activity of polypeptides of the present invention: agonists orantagonists of polypeptides in the invention), characterized bycomparative assay of the extent to which labeled polypeptides of thepresent invention bind to polypeptide receptors of the present inventionbetween cases in which labeled polypeptides of the present invention arebrought into contact with polypeptide receptors of the inventionexpressed on cell membranes through the culture of transformantscontaining DNA coding for polypeptide receptors of the presentinvention, and cases in which labeled polypeptides of the presentinvention and test compounds are brought into contact with polypeptidereceptors of the present invention expressed on cell membranes throughthe culture of transformants containing DNA coding for the polypeptidereceptors of the present invention;

(iv) methods for screening compounds or their salts which modify bindingbetween polypeptides of the present invention and polypeptide receptorsof the present invention (compounds that promote or inhibit the activityof polypeptides of the present invention: agonists or antagonists ofpolypeptides in the invention), characterized by comparative assay ofpolypeptide receptor-mediated cell-stimulating activity (such asactivity in promoting or inhibiting arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potential, intracellular proteinphosphorylation, c-fos activation, and decreases in pH) between cases inwhich compounds that activate polypeptide receptors of the presentinvention (such as polypeptides of the present invention) are broughtinto contact with cells containing polypeptide receptors of the presentinvention, and cases in which compounds that activate polypeptidereceptors of the present invention and test compounds are brought intocontact with cells containing polypeptide receptors of the presentinvention; and

(v) methods for screening compounds or their salts which modify bindingbetween polypeptides of the present invention and polypeptide receptorsof the present invention (compounds that promote or inhibit the activityof polypeptides of the present invention: agonists or antagonists ofpolypeptides in the invention), characterized by comparative assay ofpolypeptide receptor-mediated cell-stimulating activity (such asactivity in promoting or inhibiting arachidonic acid release,acetylcholine release, intracellular Ca²⁺ release, intracellular cAMPproduction, intracellular cGMP production, inositol phosphateproduction, changes in cell membrane potential, intracellular proteinphosphorylation, c-fos activation, and decreases in pH) between cases inwhich compounds that activate polypeptide receptors of the presentinvention (such as polypeptides of the present invention) are broughtinto contact with polypeptide receptors of the invention expressed oncell membranes through the culture of transformants containing DNAcoding for polypeptide receptors of the present invention, and cases inwhich compounds that activate polypeptide receptors of the presentinvention and test compounds are brought into contact with polypeptidereceptors of the present invention expressed on cell membranes throughthe culture of transformants containing DNA coding for the polypeptidereceptors of the present invention.

Screening methods of the present invention are described in detailbelow.

The polypeptide receptors of the present invention used in the screeningmethod of the invention may be any that recognize polypeptides of theinvention as ligands, although membrane fractions and the like of humanor warm-blooded animal organs are preferred. However, because of theextreme difficulties involved in procuring human organs in particular,polypeptide receptors of the present invention which have been expressedin large amounts using recombinants are suitable for use in screening.

The same methods as the aforementioned methods for producingpolypeptides of the present invention can be used to produce polypeptidereceptors of the invention.

The following methods of preparation should be followed when cells orcell membrane fractions containing polypeptide receptors of the presentinvention are used in the screening methods of the present invention.

When cells containing polypeptide receptors of the present invention areused, the cells may be immobilized with glutaraldehyde, formalin, or thelike. The cells can be immobilized in accordance with a method that iswell-known per se.

Cells containing polypeptide receptors of the present invention refer tohost cells in which such polypeptide receptors are expressed. Desirableexamples of such host cells include Escherichia coli, Bacillus subtilis,yeasts, insect cells, and animal cells. Host cells expressingpolypeptide receptors of the present invention can be obtained in thesame manner as methods for producing transformants obtained withexpression vectors containing polypeptides of the invention, asdescribed above.

Cell membrane fractions refer to fractions containing an abundance ofcell membranes, which are obtained by methods that are well-known per seafter the cells have been disrupted. Methods for disrupting cellsinclude methods for crushing cells with a Potter-Elvehjem homogenizer,disruption with a Waring blender or a Polytron (manufactured byKinematica), ultrasonic disruption, and disruption using a French pressor the like, where the cells are discharged under pressure throughnarrow nozzles. Fractions of cell membranes are obtained primarilythrough fractionation with centrifugal force, such as fractioncentrifugation or density gradient centrifugation. For example, celllysates are centrifuged for a short period of time (usually about 1 to10 minutes) at low speed (500 to 3,000 rpm), and the supernatant is thenusually further centrifuged for 30 minutes to 2 hours at high speed(15,000 to 30,000 rpm), giving membrane fractions in the form ofprecipitate. Such membrane fractions contain an abundance of theexpressed polypeptide receptors of the present invention and membranecomponents such as cell phospholipids or membrane proteins.

The amount of the polypeptide receptors of the invention contained inthe cells or membrane fractions which contain such polypeptide receptorsshould be 10³ to 10⁸ molecules, and more preferably 10⁵ to 10⁷molecules, per cell. The greater the amount expressed, the higher thepolypeptide receptor binding activity (specific activity) per membranefraction, which not only allows the construction of highly sensitivescreening systems but also allows the assay of large amounts of sampleper lot.

Suitable polypeptide receptor fractions and labeled polypeptides of thepresent invention, for example, can be used in methods (i) through (iii)above to screen for compounds that modify binding between polypeptidesof the present invention and polypeptide receptors of the presentinvention (compounds that promote or inhibit the activity ofpolypeptides in the present invention: agonists or antagonists ofpolypeptides in the invention). Preferred examples of polypeptidereceptor fractions of the present invention include native polypeptidereceptor fractions, and recombinant polypeptide receptor fractions withequivalent activity. As used herein, “equivalent activity” meansequivalent polypeptide binding activity and the like. Labeled ligands ofthe invention include both labeled ligands and labeled polypeptideanalog compounds. Examples include ligands labeled with [³H], [¹²⁵I],[¹⁴C] and [³⁵S].

Specifically, in order to screen compounds that modify binding betweenpolypeptides of the present invention and polypeptide receptors of thepresent invention, a receptor preparation can first be prepared bysuspending cells or cell membrane fractions containing polypeptidereceptors of the present invention in buffer suitable for screening.Examples of buffer include any that will not inhibit binding betweenpolypeptides of the present invention and receptors, such as Tris-HClbuffer or phosphate buffer with a pH of 4 to 10 (and preferably a pH of6 to 8). Surfactant such as CHAPS, Tween-80™ (by Kao-Atlas), digitonin,and deoxycholate can be added to the buffer to reduce non-specificbinding. A protease inhibitor such as PMSF, leupeptin, E-64(manufactured by Peptide Institute, Inc.), or pepstatin can also beadded to inhibit the degradation of polypeptides or receptors of theinvention by proteases. A certain amount (5,000 cpm to 500,000 cpm) oflabeled polypeptide in the present invention is added to 0.01 to 10 mLof the above receptor solution in the presence of 10⁻¹⁰ M to 10⁻⁷ M testcompound. A reaction tube with an excess of unlabeled polypeptide of theinvention is also prepared to determine the non-specific binding (NSB).The reaction is carried out for about 20 minutes to 24 hours, andpreferably about 30 minutes to 3 hours, at a temperature of about 0° C.to 50° C., and preferably about 40° C. to 37° C. After the reaction, thereaction mixture is filtered through glass fiber filter paper or thelike and is washed with a suitable amount of the same buffer, and theradioactivity remaining on the glass fiber filter paper is measured witha liquid scintillation counter or γ-counter. Candidates withantagonist-inhibiting capacity can be selected from test compounds witha specific binding (B-NSB) of no more than 50%, for example, where 100%is the count (B₀-NSB) calculated by subtracting the non-specific binding(NSB) from the count prevailing in the absence of any competingsubstances (B₀).

Cell-stimulating activity mediated by polypeptide receptors in thepresent invention (such as activity in promoting arachidonic acidrelease, acetylcholine release, intracellular Ca²⁺ release,intracellular cAMP production, intracellular cGMP production, inositolphosphate production, changes in cell membrane potential, intracellularprotein phosphorylation, c-fos activation, and decreases in pH) can beassayed using a common method or a commercially available kit in methods(iv) and (v) above to screen for compounds that modify binding betweenpolypeptides of the present invention and polypeptide receptors of thepresent invention (compounds that promote or inhibit the activity ofpolypeptides in the present invention: agonists and antagonists ofpolypeptides in the invention). Specifically, cells containingpolypeptide receptors of the invention are first cultured in multi-wellplates or the like. In performing the screening, the medium is replacedwith fresh medium or a suitable buffer that is not toxic to the cells,test compound or the like is added, the mixture is incubated for acertain period of time, the cells are then extracted or the supernatantis collected, and the product is quantified according to a variety ofmethods. When the production of a substance serving as an indicator ofcell-stimulating activity (such as arachidonic acid) proves difficult todetect because of degrading enzymes in the cells, the assay may beperformed with the addition of an inhibitor for such enzymes. Activitysuch as the inhibition of cAMP production can be detected in terms ofthe inhibited production of cells whose basic production is increasedwith forskolin or the like.

Cells expressing suitable polypeptide receptors of the present inventionare necessary for screening by assay of cell-stimulating activity.Preferred cells for expressing polypeptide receptors of the presentinvention include cell lines expressing such polypeptide receptors.

Examples of test compounds include peptides, proteins, non-peptidecompounds, synthetic compounds, fermented products, cell extracts, plantextracts, and animal tissue extracts.

Screening kits for compounds or their salts that modify binding betweenpolypeptides of the present invention or their precursor proteins andpolypeptide receptors of the present invention (compounds that promoteor inhibit the activity of polypeptides of the invention: agonists andantagonists of polypeptides in the invention) can include polypeptidereceptors of the present invention or their salts, partial peptides ortheir salts of polypeptide receptors of the invention, cells containingpolypeptide receptors of the present invention, cell membrane fractionscontaining polypeptide receptors of the present invention, andpolypeptides of the invention.

The following are examples of such screening kits.

1. Screening Reagents

(i) Assay Buffer and Washing Buffer

Hanks' balanced salt solution (by Gibco) supplemented with 0.05% bovineserum albumin (by Sigma).

This can be sterilized by filtration with a filter having a pore size of0.45 μm, and stored at 4° C., or it can be prepared at the time of use.

(ii) Polypeptide Receptor Preparation

CHO cells expressing polypeptide receptors of the present invention aresubcultured with 5×10⁵ cells/well in 12-well plates, and are culturedfor 2 days at 37° C. in 5% CO₂ and 95% air.

(iii) Labeled Ligands

Polypeptides of the present invention labeled with [³H], [¹²⁵I], [¹⁴C],[³⁵S] or the like.

Stored dissolved in a suitable solvent or buffer at 4° C. or −20° C.,and diluted with assay buffer to 1 μM at the time of use.

(iv) Ligand Label Solution

The polypeptides of the present invention are dissolved to aconcentration of 1 mM in PBS containing 0.1% bovine serum albumin (bySigma), and stored at −20° C.

2. Assay

(i) Cells expressing the polypeptide receptors of the present invention,which have been cultured in 12-well tissue culture plates, are washedtwice with 1 mL assay buffer, and 490 μL assay buffer is then added perwell.

(ii) 5 μL of 10⁻³ to 10⁻¹⁰ M test compound solution is added, 5 μLlabeled polypeptide of the invention is then added, and a reaction isbrought about for 1 hour at ambient temperature. 5 μL of 10⁻³ Mpolypeptide of the invention is added instead of test compound todetermine the non-specific binding.

(iii) The reaction solution is removed, and the cells are washed 3 timeswith 1 mL washing buffer. The labeled polypeptide of the inventionbinding to the cells is dissolved in 0.2 N NaOH-1% SDS and mixed with 4mL liquid Scintillator A (Wako Pure Chemicals).

(iv) The radioactivity is assayed using a liquid scintillation counter(Beckman), and the percent maximum binding (PMB) is determined using thefollowing equation.PMB={(B-NSB)/(B ₀-NSB)}×100PMB: percent maximum bindingB: value when sample addedNSB: non-specific bindingB₀: maximum binding  {Equation 1}

Compounds or their salts obtained using the screening methods orscreening kits of the invention are compounds that modify (promote orinhibit) binding between polypeptides of the invention and polypeptidereceptors of the invention (compounds that promote or inhibit theactivity of polypeptides of the invention: agonists and antagonists ofthe invention), specifically, compounds or their salts withcell-stimulating activity mediated by polypeptide receptors of thepresent invention (referred to as polypeptide agonists of theinvention), or compounds with no such cell-stimulating activity(referred to as polypeptide antagonists of the invention). Examples ofsuch compounds include peptides, proteins, non-peptide compounds,synthetic compounds, and fermented products. Such compounds may be novelcompounds or known compounds.

The following specific methods (i) or (ii) should be followed toevaluate whether the compounds are agonists or antagonists of thepolypeptides of the present invention.

(i) Binding assay is performed as indicated in the screening methods of(i) through (iii) above to obtain compounds that modify (and inhibit, inparticular) binding between polypeptides of the invention andpolypeptide receptors of the invention, and it is then determinedwhether or not the compounds have cell-stimulating activity mediated bythe aforementioned polypeptide receptors of the invention. Compounds orsalts with cell-stimulating activity are polypeptide agonists of theinvention, while compounds or salts with no such activity arepolypeptide antagonists of the invention.

(ii) (a) Test compounds are brought into contact with cells containingpolypeptide receptors of the invention to assay the cell-stimulatingactivity mediated by the aforementioned polypeptide receptors of theinvention. Compounds or their salts with cell-stimulating activity arepolypeptide agonists of the invention.

(b) Cell-stimulating activity mediated by polypeptide receptors of theinvention is comparatively assayed between cases in which compounds thatactivate polypeptide receptors of the invention (such as polypeptides ofthe invention or agonists of polypeptide in the invention) are broughtinto contact with cells containing polypeptide receptors of theinvention, and cases in which test compounds and compounds that activatepolypeptide receptors of the invention are brought into contact withcells containing polypeptide receptors of the invention. Compounds ortheir salts that are capable of reducing the cell-stimulating activitycaused by compounds that activate polypeptide receptors of the inventionare polypeptide antagonists of the invention.

Polypeptide agonists of the invention have action similar to thephysiological activity of polypeptides of the invention on polypeptidereceptors of the invention, and are therefore useful as safe drugs withlow toxicity in the same manner as polypeptides of the invention (suchas drugs for the treatment and/or prevention of blood pressure disorders(such as hypertension), exocrine disorders, endocrine disorders, lipiddysmetabolism, and cardiovascular diseases).

Conversely, polypeptide agonists of the invention are capable ofinhibiting the physiological activity of polypeptides of the inventionon polypeptide receptors of the invention, and are therefore useful assafe drugs with low toxicity in inhibiting such receptor activity (suchas drugs for the treatment and/or prevention of allergic diseases,asthma, angina pectoris, atherosclerosis, diabetes, hyperlipemia,emesis, bone diseases, pollakiuria, AIDS, and blood pressure disorders).

Compounds or their salts obtained using the screening methods orscreening kits of the invention can be selected from peptides, proteins,non-peptide compounds, synthetic compounds, fermented products, cellextracts, plant extracts, animal tissue extracts, plasma, or the like,and are compounds that promote or inhibit functions of the polypeptidesof the invention.

Examples of salts of such compounds are the same as the examples ofsalts given for polypeptides of the present invention above.

Common procedures can be followed when compounds obtained using thescreening methods or kits of the invention are used as the remedies andprophylactics described above. For example, they can be used in the formof tablets, capsules, elixirs, microcapsules, sterile solutions,suspensions, or the like in the same manner as drugs containingpolypeptides of the invention, as described above.

Because such preparations are safe and have low toxicity, they can beadministered orally and parenterally, for example, to humans andwarm-blooded animals (such as mice, rats, rabbits, sheep, pigs, cows,horses, birds, cats, dogs, monkeys, and chimpanzees).

The dosage of such compounds and salts will vary depending on theiractivity, the target disease, purpose of administration, route ofadministration or the like, but the daily adult oral dosage of compoundsthat promote the functions of polypeptides of the invention for thetreatment of hypertensions, for example, may generally range from about0.1 to 100 mg, preferably from about 1.0 to 50 mg, and even morepreferably from about 1.0 to 20 mg, in terms of compound (per 60 kg bodyweight). The single parenteral dose of such compounds will varydepending on the purpose of administration, target disease, and thelike, but in the form of an injection for adults, for example, the dailydosage of compounds that promote the functions of polypeptides of theinvention for treatment of hypertension may usually range from about0.01 to about 30 mg, preferably about 0.1 to about 20 mg, and even morepreferably about 0.1 to about 10 mg at a time, in terms of compound (per60 kg body weight), given by intravenous injection. Doses for animalscan be given as calculated per 60 kg body weight.

(3) Quantification of Polypeptides of the Invention

Antibodies against polypeptides of the invention (sometimes referred tobelow simply as antibodies of the invention) specifically recognizepolypeptides of the invention, and can therefore be used to quantifypolypeptides of the invention in analyte, particularly assay by sandwichimmunoassay.

Specifically, the invention is intended to provide:

(i) a method for quantifying polypeptides of the invention in analyte,characterized by conducting a competitive reaction of antibody of theinvention with analyte and labeled polypeptides of the invention todetermine the proportion of labeled polypeptides of the inventionbinding to the antibody; and

(ii) a method for quantifying polypeptides of the invention in analyte,characterized by conducting simultaneous or continuous reaction ofanalyte with antibody of the present invention insolubilized on acarrier and other labeled antibody of the invention, and then assayingthe activity of the label on the insolubilization carrier.

In the method of quantification in (ii), one antibody should recognizethe N terminal of polypeptides of the invention, and the other antibodyshould react with the C terminal of polypeptides of the invention.

Polypeptides of the invention can be quantified using monoclonalantibodies against polypeptides of the invention, but they can also bedetected by tissue staining, or the like. For that purpose, the antibodymolecules themselves may be used, and F(ab′)₂, Fab′, or Fab fractions ofantibody molecules may also be used.

Quantification of polypeptides of the present invention using antibodiesof the invention is not particularly limited. Any method ofquantification can be used in which the amount of antibody, antigen, orantibody-antigen complex relative to the amount of antigen (such as theamount of polypeptide) in the analyte is detected by chemical orphysical means, and is calculated from a standard curve prepared using astandard containing a known amount of antigen. For example, nephrometry,competitive methods, immunometric methods, and sandwich methods aresuitable for use, although the use of a sandwich method, as describedbelow, is preferred in terms of sensitivity and specificity.

Examples of labels for use in assays using labeled substances includeradioisotopes, enzymes, fluorescent substances, and luminescentsubstances. Examples of radioisotopes include [¹²⁵I], [¹³¹I], [³H] and[¹⁴C]. Examples of enzymes include those that are stable and that havehigh specific activity, such as β-galactosidase, β-glucosidase, alkaliphosphatase, peroxidase, and malate dehydrogenase. Examples offluorescent substances include fluorescamine and fluoresceinisothiocyanate. Examples of luminescent substances include luminol,luminol derivatives, luciferin, and lucigenin. A biotin-avidin systemmay also be used for binding between antibody or antigen and a label.

Physical adsorption may be employed for the insolubilization of antigensor antibodies. Methods employing chemical bonding may also normally beused for the insolubilization or immobilization of polypeptides,enzymes, or the like. Examples of carriers include insolublepolysaccharides such as agarose, dextran and cellulose, synthetic resinssuch as polystyrene, polyacrylamide and silicone, and glass or the like.

In a sandwich method, the test liquid is allowed to react withinsolubilized monoclonal antibody of the present invention (primaryreaction), separate labeled monoclonal antibody of the present inventionis allowed to react (secondary reaction), and the activity of the labelon the insoluble carrier is then assayed so as to quantify the amount ofpolypeptides of the present invention in the analyte. The primary andsecondary reactions may be carried out in reverse order, simultaneously,or while staggered. The label and method of insolubilization can bebased on those described above. In sandwich immunoassay, the antibodyused for the labeled antibody or solid phase antibody need notnecessarily be one type; a mixture of two or more types of antibody maybe used to improve assay sensitivity or the like.

The monoclonal antibodies of the present invention which are used in theprimary and secondary reactions in the sandwich assay of polypeptides ofthe invention should have different binding sites for polypeptides ofthe invention. Specifically, the antibodies used in the primary reactionshould recognize a region other than the C terminal region, such as theN terminal region, for example, whenever the antibody used in thesecondary reaction recognizes the C terminal region of polypeptides inthe invention.

Monoclonal antibodies of the present invention may be used in assaysystems other than sandwich assay, such as competitive methods,immunometric methods, and nephrometry.

In competitive methods, labeled antigen and antigen in an analyte areallowed to undergo competitive reaction with antibody, the unreactedlabeled antigen (F) and the labeled antigen (B) binding to the antibodyare then separated (B/F separation), and the amount of label in either Bor F is determined so as to quantify the amount of antigen in theanalyte. This method of reaction can entail the use of a liquid phasemethod in which soluble antibody is used as the antibody. Polyethyleneglycol and secondary antibody against the aforementioned antibody areused in the B/F separation. A solid phase method in which immobilizedantibody is used as primary antibody, or the primary antibody is solubleand immobilized antibody is used as the secondary antibody.

In immunometric methods, immobilized antigen and antigen in analyte areallowed to undergo competitive reaction with a given amount of labeledantibody. The solid and liquid phases are then separated, or the antigenin the analyte is allowed to react with an excess of labeled antibody.The immobilized antigen is then added to allow the unreacted labeledantibody bind to the solid phase. The solid and liquid phases are thenseparated. The amount of label in either phase is then determined toquantify the amount of antigen in the analyte.

In nephrometry, the amount of insoluble precipitate produced as a resultof an antigen-antibody reaction in gel or solution is measured. Lasernephrometry based on laser scattering or the like is suitable for use incases involving trace amounts of antigen in analyte which result in onlyminute amounts of precipitate.

No special conditions, operations or the like need to be established inorder to apply these individual immunoassay methods to thequantification method of the present invention. The polypeptide assaysystem of the present invention should be constructed based on commontechnical considerations known to those having ordinary skill in the artfor the usual conditions and operations in the individual methods above.The general technical details can be found in references, documents, andthe like.

Examples include Hiroshi Irie, Ed., Radioimmunoassay (published byKodansha (1974)); Hiroshi Irie, Ed., Radioimmunoassay, Part II(published by Kodansha (1979)); Eiji Ishikawa et al. , Ed., EnzymeImmunoassay (published by Igaku Shoin (1978)); Eiji Ishikawa et al.,Ed., Enzyme Immunoassay (Second Edition) (published by Igaku Shoin(1982)); Eiji Ishikawa et al., Ed., Enzyme Immunoassay (Third Edition)(published by Igaku Shoin (1987)); Methods in Enzymology Vol. 70(Immunochemical Techniques (Part A)); ibid. Vol. 73 (ImmunochemicalTechniques (Part B)); ibid. Vol. 74 (Immunochemical Techniques (PartC)); ibid. Vol. 84 (Immunochemical Techniques (Part D: SelectedImmunoassays)); ibid. Vol. 92 (Immunochemical Techniques (Part E:Monoclonal Antibodies and General Immunoassay Methods)); ibid. Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (Academic Press).

Antibodies of the present invention can be used in the manner describedabove for the sensitive quantification of polypeptides of the presentinvention.

Decreases or increases in the concentration of polypeptides of theinvention can be detected through the quantification of theconcentration of polypeptides of the present invention using antibodiesof the present invention in order to permit the diagnosis of thepresence or the high possibility of future onset of diseases such asblood pressure disorders (such as hypertension), exocrine disorders, andcardiovascular diseases.

The antibodies of the invention can also be used to detect polypeptidesof the invention present in analytes such as bodily fluids and tissue.They can also be used to prepare antibody columns for use in thepurification of polypeptides of the invention, to detect polypeptides ofthe invention in fractions during purification, to analyze the behaviorof polypeptides of the invention in analyte cells, and so forth.

(4) Genetic Diagnostic Agents

Polynucleotides (DNA) of the present invention can be used as probe, forexample, to detect abnormalities (genetic abnormalities) in DNA or mRNAcoding for polypeptides of the invention in humans or warm-bloodedanimals (such as rats, mice, guinea pigs, rabbits, birds, sheep, pigs,cows, horses, cats, dogs, and monkeys), and can thus be useful asgenetic diagnostic agents for ascertaining damage to, variation in, orunder-expression of DNA or mRNA, or increases in or over-expression ofDNA or mRNA.

Such genetic diagnostics using DNA of the invention can be managed bymethods that are known per se, such as Northern hybridization orPCR-SSCP (Genomics, Vol. 5, 874-879 (1989); and Proceedings of theNational Academy of Sciences of the United States of America, Vol. 86,2766-2770 (1989)).

Under-expression revealed by Northern hybridization can permit thediagnosis of the presence or the possibility of future onset of diseasessuch as blood pressure disorders (hypertension), exocrine disorders, orcardiovascular diseases.

Conversely, over-expression revealed by Northern hybridization canpermit the diagnosis of the presence or the possibility of future onsetof diseases such as allergic diseases, asthma, angina pectoris,atherosclerosis, diabetes, hyperlipemia, emesis, bone diseases,pollakiuria, AIDS, and blood pressure disorders.

The nucleotide (DNA) sequences of the invention are also useful foridentifying the chromosomes of organisms. Hybridization with certainlocations on the chromosomes of a target organism can allow chromosomesrelated to genes of the invention to be identified and mapped. Genesinvolved in diseases associated with polypeptides of the invention canthus be identified by such mapping.

When polynucleotides (DNA) of the invention are used as tools toidentify genes involved in diseases associated with polypeptides of theinvention, the use of polynucleotides (DNA) containing base sequencescoding for ATT, ATTshort1, and ATTshort2 is particularly preferred,although the use of polynucleotides (DNA) containing a base sequencecoding for an amino acid sequence that included the C terminal amidationconsensus (Gly-Lys-Arg) (ATT-Gly-Lys-Arg, ATTshort1-Gly-Lys-Arg, andATTshort2-Gly-Lys-Arg) is even more preferable.

Cells from organisms having variation or diversity (or allele variation)among the polynucleotides and/or polypeptides of the invention can bedetected at the level of DNA by various techniques which permitserotyping, for example. Variation in RNA can be detected using RT-PCR,for example. RT-PCR should especially be used in combination with anautomatic detection system such as a commercially available imageanalyzer. RNA, cDNA, or genomic DNA can all be used in PCR or RT-PCR forthe same purpose. An example is the ability to identify and analyzevariation using PCR primers complementary to the nucleic acids codingfor polypeptides of the invention.

(5) Drugs Containing Antisense DNA

Antisense DNA capable of complementarily binding to DNA of the presentinvention to inhibit the expression of such DNA can be used as an agentin the treatment and/or prevention of diseases such as allergicdiseases, asthma, angina pectoris, atherosclerosis, diabetes,hyperlipemia, emesis, bone diseases, pollakiuria, AIDS, and bloodpressure disorders.

In such antisense DNA applications, the antisense DNA can be usedaccording to common methods, either by itself or after beingincorporated into suitable vectors such as retrovirus vectors,adenovirus vectors, and adenovirus-associated virus vectors. Suchantisense DNA can be given, either as such or in the form of apreparation combined with a physiologically acceptable carrier such asan adjuvant to facilitate ingestion, by means of a gene gun or acatheter such as a hydrogel catheter.

Antisense DNA can also be used as a diagnostic oligonucleotide probe tocheck for the presence or the expression of DNA of the present inventionin tissue or cells.

(6) Drugs Containing Antibodies of the Invention

Antibodies of the invention which have activity to neutralizepolypeptides of the invention can be used as drugs for the treatmentand/or prevention of diseases such as allergic diseases, asthma, anginapectoris, atherosclerosis, diabetes, hyperlipemia, emesis, bonediseases, pollakiuria, AIDS, and blood pressure disorders.

Such agents containing antibodies of the present invention for thetreatment and/or prevention of the aforementioned diseases can be givenorally or parenterally in unmodified liquid form or in the form ofsuitable pharmaceutical compositions to humans or mammals (such as rats,rabbits, sheep, pigs, cows, cats, dogs, and monkeys). The dosage of willvary depending on the purpose of administration, target disease,symptoms, route of administration or the like, but the single adultdosage of antibodies of the present invention for the treatment ofallergic diseases, for example, may generally range from about 0.01 to20 mg/kg, preferably from about 0.1 to 10 mg/kg, and even morepreferably from about 0.1 to 5 mg, to be given by intravenous injectionabout 1 to 5 times a day, and preferably about 1 to 3 times a day. Adosage based on this can be given for other types of parenteraladministration or oral administration. Doses based on this can be givenin other form so parenteral administration and oral administration. Thedosage may be increased if symptoms are particularly serious.

Antibodies of the invention can be given as such or in the form ofsuitable pharmaceutical compositions. Pharmaceutical compositions usedfor the administration described above include the above or their saltswith pharmaceutically acceptable carriers, diluents, or excipients. Suchcompositions may be provided in the form of preparations suitable fororal or parenteral administration.

That is, they can be used in the form of compositions for oraladministration, specifically, tablets (including sugar-coated tabletsand film-coated tablets), pills, granules, dispersions, capsules(including soft capsules), syrups, emulsions, and suspensions. Suchcompositions can be produced by methods that are known per se, and caninclude carriers, diluents, or excipients commonly used in thepharmaceutical field. Examples of carriers and excipients for tabletsinclude lactose, starch, sucrose, and magnesium stearate.

Examples of compositions for parenteral use include injections, andsuppositories. Injections include intravenous injections, subcutaneousinjections, intracutaneous injections, intramuscular injections, anddrip infusions. Such injections can be produced in accordance withmethods that are known per se, such as by dissolving, suspending, oremulsifying the aforementioned antibodies or their salts in sterileaqueous or oleaginous liquids commonly used in injections. Aqueousliquids for injection include physiological saline and isotonicsolutions containing glucose or other adjuvants, and may be used incombination with appropriate dissolution aids such as alcohols (such asethanol), polyalcohols (such as propylene glycol and polyethyleneglycol), and nonionic surfactants (such as Polysorbate 80™ and HCO-50(polyoxyethylene (50 mol) adduct of hydrogenated castor oil)).Oleaginous liquids include sesame oil and soybean oil, and may be usedin combination with benzyl benzoate, benzyl alcohol, and the like asdissolution aids. Suitable ampules are usually aseptically filled withthe resulting injection liquid. Suppositories for rectal administrationmay be prepared by mixing the aforementioned antibodies or salts with acommon suppository base.

The aforementioned oral and parenteral pharmaceutical compositions maybe prepared in the unit dose forms suitable for the dosage of activeingredient. Such unit dose forms include tablets, pills, capsules,injections (ampules), and suppositories, and should usually containantibody in an amount of 5 to 500 mg per unit dose form, and morepreferably 5 to 100 mg in injections and 10 to 250 mg in otherformulations.

The aforementioned compositions may contain other active ingredients,provided that their combination with the aforementioned antibodies doesnot result in any undesirable interaction.

(7) Transgenic Animals

The invention is also intended to provide non-human mammals withexogenous DNA coding for polypeptides of the present invention(sometimes referred to below simply as exogenous DNA) or mutant DNAthereof (sometimes referred to below simply as exogenous mutant DNA).

Specifically, the present invention is intended to provide:

(1) non-human mammals with exogenous DNA of the present invention ormutant DNA thereof;

(2) animals as described in (1) above, wherein the non-human mammal is arodent;

(3) animals as described in (2) above, wherein the rodent is a mouse orrat; and

(4) recombinant vectors which contain exogenous DNA or mutant DNA of thepresent invention, and which are capable of expression in mammals.

Non-human mammals with exogenous DNA or mutant DNA thereof in theinvention (referred to below as transgenic animals of the invention) canbe prepared by introducing the target DNA by means of calcium phosphate,electroporation, lipofection, agglutination, microinjection, particlegun, or DEAE-dextran or the like into germinal cells or the like,including fertilized or unfertilized eggs, or spermatozoa or theirprimordial cells, preferably at the embryonic stage (and more preferablyat the single cell or fertilized egg cell stage, or generally within the8-cell stage) in the development of non-human mammals. Such methods forintroducing DNA can be employed to introduce target exogenous DNA of theinvention into somatic cells, the organs of organisms, tissue cells, orthe like for use in cell culture, tissue culture, or the like. The cellscan be fused by common methods of cell fusion with the aforementionedgerminal cells to create transgenic animals of the invention.

Examples of non-human mammals include cows, pigs, sheep, goats, rabbits,dogs, cats, guinea pigs, hamsters, mice, and rats. Animals that arepreferred among these for the purposes of preparing disease animalmodels include rodents, which are characterized by relatively rapidontogeny and life cycle, and are easy to breed, particularly mice (suchas pure strains like C57BL/6 and DBA2, and hybrid strains like B6C3F₁,BDF₁, B6D2F₁, BALB/c, and ICR) or rats (such as Wistar and SD).

“Mammals,” as used in the context of recombinant vectors capable ofexpression in mammals, include the non-human mammals noted above, aswell as humans.

The exogenous DNA of the present invention refers not to DNA of thepresent invention which is inherent to non-human mammals, but refers toDNA of the present invention which has been isolated and extracted frommammals.

Examples of variant DNA in the present invention include that producedby variations (such as mutations) in the base sequence of the originalDNA in the present invention, specifically, DNA with added or deletedbases, substitutions with other bases, or the like, as well as abnormalDNA.

Abnormal DNA means DNA causing the expression of abnormal polypeptidesin the present invention, such as DNA causing the expression ofpolypeptides inhibiting the function of normal polypeptides of theinvention.

Exogenous DNA of the invention may be from a mammal of either the sameor different species of the target animal. To introduce DNA of theinvention into a target animal, it is generally beneficial to use DNA inthe form of a DNA construct which is ligated downstream of a promoterthat enables expression of the DNA in animal cells. During theintroduction of the human DNA of the present invention, for example, DNAconstructs (such as vectors) which comprise the human DNA of the presentinvention ligated downstream of various promoters that enable expressionof the DNA of various mammals (such as rabbits, dogs, cats, guinea pigs,hamsters, rats, and mice) that have DNA of the present inventionpossessing high homology with the above can be microinjected tofertilized eggs of the target mammals, such as mouse fertilized eggs, soas to construct a transgenic mammal with high expression of DNA in thepresent invention.

Examples of expression vectors for polypeptides of the invention includeE. coli plasmids, B. subtilis plasmids, yeast plasmids, λ-phages andother bacteriophages, retroviruses such as Moloney leukemia virus, andanimal viruses such as vaccinia virus and baculovirus. Preferredplasmids include E. coli plasmids, B. subtilis plasmids, and yeastplasmids.

Examples of promoters that regulate the expression of such DNA include(i) virus (such as simian virus, cytomegalovirus, Moloney leukemiavirus, JC virus, papilloma virus, and poliovirus) DNA promoters; (ii)mammal (such as human, rabbit, dog, cat, guinea pig, hamster, rat, andmouse) promoters, such as albumin, insulin II, uroplakin II, elastase,erythropoietin, endothelin, muscle creatine kinase, glial fibrillaryacidic protein, glutathione S-transferase, platelet-derived growthfactor β, keratin K1, K10, and K14, collagen type I and type II, cyclicAMP-dependent protein kinase βI subunit, dystrophin, tartaricacid-resistant alkaline phosphatase, atrial natriuretic factor,endothelial receptor tyrosine kinase (commonly abbreviated as Tie2),sodium/potassium-exchanging adenosine triphosphatase (Na, K-ATPase),neurofilament light chain, metallothionein I and IIA, metalloprotease Itissue inhibitor, MHC Class I antigen (H-2L), H-ras, renin, dopamineβ-hydroxylase, thyroid peroxidase (TPO), polypeptide chain elongationfactor 1α (EF-1α), β actin, α and β-myosin heavy chain, myosin lightchains 1 and 2, myelin basic protein, thyroglobulin, Thy-1,immunoglobulin, H chain variable region (VNP), serum amyloid Pcomponent, myoglobin, troponin C, smooth muscle α-actin,preproenkephalin A, and vasopressin. Preferable promoters are promotersconducive to high expression throughout the entire body, such ascytomegalovirus promoter, human polypeptide chain elongation factor 1α(EF-1α) promoter, and human and chicken β-actin promoters.

The aforementioned vectors should have a sequence for terminating thetranscription of the target mRNA (generally called the terminator) inthe transgenic mammal, examples of which include DNA sequences ofviruses and various mammals, preferably simian virus SV40 promoter orthe like.

A splicing signal, enhancer region, a portion of eukaryotic DNA intron,or the like can be ligated upstream of the 5′-end of the promoterregion, between the promoter region and the translated region, ordownstream of the 3′-end of the translated region, depending on thepurpose, in order to ensure higher expression of the target exogenousDNA.

The translated region of normal polypeptides of the present inventioncan be obtained in the form of either all or part of genomic DNA from avariety of commercially available genomic DNA libraries and DNA from theliver, kidneys, thyroid cells, or fibroblasts of humans or variousmammals (such as rabbits, dogs, cats, guinea pigs, hamsters, rats, andmice), or by using as starting material complementary DNA prepared by acommon method from RNA of liver, kidneys, thyroid cells or fibroblasts.Exogenous abnormal DNA can be obtained by preparing translated regionsin which point mutations have been induced in the translated region ofnormal polypeptides obtained from the aforementioned cells or tissue.

The translated region can be prepared by common DNA engineering methodsin which a DNA construct capable of expression in transgenic animals isligated downstream of the aforementioned promoter such as the above andupstream of the transcription termination site as desired.

The introduction of exogenous DNA of the present invention into thefertilized egg cell stage can be managed in such a way as to ensure itspresence in all germinal cells and somatic cells of the target mammal.The presence of exogenous DNA of the invention in the germinal cells ofthe transgenic animal means that all offspring of the transgenic animalwill have the exogenous DNA of the invention in all their germinal cellsand somatic cells. The offspring of animals of this line inheriting theexogenous DNA of the invention will have the exogenous DNA of thepresent invention in all their germinal cells and somatic cells.

Non-human mammals with the normal exogenous DNA of the present inventioncan be mated to verify stable retention of the exogenous DNA, and can bebred and raised in the usual breeding environment as animals conservingthe DNA.

The introduction of exogenous DNA of the invention into the fertilizedoocyte stage can be managed in such a way as to ensure its excesspresence in all germinal cells and somatic cells of the target mammal.The excess presence of exogenous DNA of the invention in the germinalcells of the transgenic animal means that all the offspring of thetransgenic animal will have an excess of the exogenous DNA of theinvention in all their germinal cells and somatic cells. The offspringof animals of this line inheriting the exogenous DNA of the inventionwill have an excess of the exogenous DNA of the present invention in alltheir germinal cells and somatic cells.

Homozygous animals having the DNA in both homologous chromosomes can bemated and bred in such a way as to ensure all offspring have an excessof such DNA.

Non-human mammals with normal DNA of the present invention arecharacterized by high expression of the normal DNA, and may ultimatelydevelop disorders involving polypeptide hyperfunction as a result of thepromotion of the function of normal endogenous DNA, making them usefulas disease model animals. For example, normal transgenic animals of thepresent invention can be used to elucidate the mechanisms of disordersassociated with polypeptides of the invention or hyperfunction ofpolypeptides of the invention, and to study methods for treating suchdiseases.

Mammals with normal exogenous DNA of the invention may also haveexacerbated symptoms associated with free polypeptides of the invention,and can thus be used to screen drugs for the treatment of diseasesassociated with polypeptides of the invention.

Non-human mammals with abnormal exogenous DNA of the present invention,meanwhile, can be mated to verify stable retention of the exogenous DNA,and can be bred and raised in the usual breeding environment as animalsconserving the DNA. The target exogenous DNA can also be incorporated inthe aforementioned plasmids for use as starting material. DNA constructswith promoters can be prepared by common DNA engineering techniques. Theintroduction of abnormal DNA of the invention into the fertilized oocytestage can be managed in such a way as to ensure its presence in allgerminal cells and somatic cells of the target mammal. The presence ofabnormal DNA of the invention in the germinal cells of the transgenicanimal means that all offspring of the transgenic animal will have theabnormal DNA of the invention in all their germinal cells and somaticcells. The offspring of animals of this line inheriting the exogenousDNA of the invention will have the abnormal DNA of the present inventionin all their germinal cells and somatic cells. Homozygous animals ofboth sexes having the DNA in both homologous chromosomes can be matedand bred in such a way as to ensure all offspring have the DNA.

Non-human mammals with abnormal DNA of the present invention arecharacterized by high expression of the abnormal DNA, and may ultimatelydevelop disorders involving functional inactivation of polypeptides ofthe present invention as a result of the inhibition of the function ofnormal endogenous DNA, making them useful as disease model animals. Forexample, transgenic animals with abnormal DNA of the present inventioncan be used to elucidate the mechanisms of disorders involvingfunctional inactivation of the polypeptide of the invention, and tostudy methods for treating such disorders.

As one specific potential use, animals with high expression of theabnormal DNA of the present invention can be used as a model forelucidating the functional inhibition of the normal polypeptide by anabnormal polypeptide of the present invention (dominant negative effect)in disorders involving functional inactivation of polypeptides of thepresent invention.

Mammals with abnormal exogenous DNA of the invention may also haveexacerbated symptoms associated with free polypeptides of the invention,and can thus be used to screen drugs for the treatment of disordersinvolving functional inactivation of polypeptides of the invention.

Examples of other potential uses of the above two types of transgenicanimals include:

(1) their use as sources of cells for tissue culture;

(2) analysis of the relationship to polypeptides which are specificallyexpressed or activated by polypeptides of the invention, based on directanalysis of DNA or RNA in tissue of transgenic mammals, or analysis ofthe composition of polypeptides expressed by the DNA;

(3) culture of cells from tissue containing the DNA by standard tissueculturing techniques for use in research on the function of tissue cellsnot generally amenable to culture;

(4) Screening of drugs which enhance cell function by using cellsdescribed in (3) above; and

(5) the isolation and purification of variant polypeptides in thepresent invention, and the preparation of their antibodies.

Transgenic animals of the present invention can also be used to studythe clinical symptoms of diseases associated with polypeptides of theinvention, including disorders involving functional inactivation ofpolypeptides of the invention, in order to obtain more detailedpathological findings in various organs in models of diseases associatedwith polypeptides of the invention, with the potential for contributingto the development of novel methods of treatment, as well as research onand treatment of secondary diseases caused by such diseases.

Furthermore, various organs can be excised from transgenic animals ofthe invention, homogenized, and treated with a proteolytic enzyme suchas trypsin to obtain free transgenic cells which can be cultured or usedto establish a cell line of cultured cells. Such materials make usefulresearch materials for studying polypeptides of the invention andelucidating their activity, such as the characterization of cellsproducing polypeptides of the invention, and the study of theirrelationship to apoptosis, differentiation, and proliferation, as wellas the mechanism of their signal transduction and abnormalities thereof.

The aforementioned testing methods, quantification methods, and the likecan also be used to provide a method for efficient and rapid screeningof drugs for such diseases in order to develop drugs for the treatmentof diseases associated with polypeptides of the invention, includingdisorders involving functional inactivation of polypeptides of theinvention, using transgenic animals of the invention. Transgenic animalsof the invention or exogenous DNA expression vectors of the presentinvention can also be used to study and develop DNA therapy for diseasesassociated with polypeptides of the invention.

(8) Knockout Animals

The present invention is also intended to provide non-human mammalembryonic stem cells in which DNA of the present invention has beeninactivated, and non-human mammals with deficient expression of DNA inthe invention.

Specifically, the invention is intended to provide:

(1) non-human mammal embryonic stem cells in which DNA of the presentinvention has been inactivated;

(2) embryonic stem cells according to (1) above, wherein the DNA isinactivated through the introduction of a reporter gene (such as the E.coli β-galactosidase gene);

(3) embryonic stem cells according to (1) above, which areneomycin-resistant;

(4) embryonic stem cells according to (1) above, wherein the non-humanmammal is a rodent;

(5) embryonic stem cells according to (4) above, wherein the rodent is amouse;

(6) non-human mammals with deficient expression of DNA in the invention,wherein the DNA of the invention has been inactivated;

(7) non-human mammals according to (6) above, wherein the DNA isinactivated through the introduction of a reporter gene (such as the E.coli β-galactosidase gene), and the reporter gene can be expressed underthe control of a promoter for the DNA of the present invention;

(8) non-human mammals according to (6) above, wherein the non-humanmammal is a rodent;

(9) non-human mammals according to (8) above, wherein the rodent is amouse; and

(10) a method for screening compounds or their salts which promote orinhibit promoter activity on the DNA of the present invention,characterized by the administration of a test compound to an mammalaccording to (7) above to search for expression of the reporter gene.

Non-human mammal embryonic stem cells in which DNA of the invention hasbeen inactivated refer to non-human mammal embryonic stem cells (EScells) in which the DNA expression capacity has been inhibited throughthe artificial addition of mutations to DNA of the invention possessedby such non-human mammals, or such stem cells in which DNA issubstantially deprived of the capacity to express polypeptides of theinvention as a result of the substantial loss of the activity ofpolypeptides of the invention encoded by the DNA (sometimes referred tobelow as knockout DNA of the invention).

The same non-human mammals described above can be used.

Examples of methods for artificially introducing mutations to DNA of thepresent invention include the deletion of some or all of a DNA sequence,or the insertion or substitution of other DNA, by genetic engineeringtechniques. Such mutations should be used, for example, to shift thecodon reading frame or disrupt promoter or exon functions in order toproduce knockout DNA of the invention.

Specific examples of non-human mammal embryonic stem cells in which DNAof the invention has been inactivated (referred to below asDNA-inactivated ES cells of the invention or knockout ES cells of theinvention) can be obtained by isolating target DNA of the inventionpossessed by non-human mammals, inserting a drug resistance gene, suchas the neomycin resistance gene or hygromycin resistance gene, or areporter gene, such as lacZ (β-galactosidase gene) or cat(chloramphenicol acetyl transferase gene), into the exon portions todisrupt the exon function, or inserting a DNA sequence that terminatesgene transcription (such as a polyA linker signal) between exons todisable synthesis of complete mRNA, inserting the resulting DNA strandhaving the DNA sequence thus constructed to disrupt the gene (referredto below as targeting vector) into the chromosomes of an animal byhomologous recombination, for example, and analyzing the resulting EScells by Southern hybridization using probe comprising a DNA sequence onor near the DNA of the invention or by PCR using primers comprising theDNA sequence on the targeting vector and the DNA sequence of anotherregion near the DNA of the invention used to produce the targetingvector, so as to screen for knockout ES cells of the invention.

Examples of original ES cells in which the DNA of the invention is to beinactivated by homologous recombination or the like include those whichhave already been established such as the above and new linesestablished according to the known method of Evans and Kaufman. Forexample, ES cells of the 129 line are generally used at present in thecase of mouse ES cells, but since the immunological background is notvery well known, it can be more beneficial to use lines which have beenestablished using C57BL/6 or BDF₁ mice (F₁ of C57BL/6 and DBA/2), astrain obtained by improving the low fertility of the C57BL/6 breedthrough hybridization with DBA/2, for example, in order to obtain EScells which are from a pure line and have a known immunologicalbackground. In addition to the advantages of fertility and healthy eggs,BDF₁ mice have the background of C57/BL/6 mice, so a benefit of ES cellsobtained using them is that the immunological background can beconverted to that of C57BL/6 mice by being back-crossed with C57BL/6mice when producing disease model mice.

Blastocysts are commonly used 3.5 days after fertilization whenestablishing an ES cell line, but large numbers of early embryos canotherwise be efficiently obtained by culturing 8-cell stage embryosuntil the blastocyst stage.

Although ES cells of either sex may be used, male ES cells are usuallymore convenient for producing germ line chimeras. The sexes should alsobe distinguished as soon as possible in order to minimize the complexityof the culture procedures.

An example of a method for sexing ES cells is to amplify and detect thesex-determining region on the Y chromosome by PCR. Approximately 10⁶cells are required in conventional karyotype analysis, whereas onlyabout 1 colony of ES cells (about 50) is needed in this method. Thissexing method thus permits the primary selection of ES cells in theinitial stages of culture, and also allows male cells to be selected atan early stage, considerably simplifying the early stages of culture.

Secondary selection can be carried out through the verification of thenumber of chromosomes by G-banding, for example. The number ofchromosomes of the resulting ES cells should be 100% of the normalnumber, but in cases where this is complicated by the physicaloperations or the like involved in establishing a line, the gene of theES cell should be knocked out and recloned to normal cells (such ascells with a chromosome number 2n=40 in mice).

The embryonic stem cell line thus established is generally characterizedby extremely good growth, but must be subcultured with extreme carebecause the ontogenic capacity tends to be lost. For example, the cellline should be cultured on suitable feeder cells such as STO fibroblastsin the presence of LIF (1 to 10,000 U/ml) in a carbon dioxide culturevessel (preferably 5% CO₂ and 95% air, or 5% oxygen, 5% CO₂, and 90%air) at about 37° C. During subculture, the cells should be treated, forexample, with trypsin/EDTA solution (usually 0.001 to 0.5% trypsin/0.1to 5 mM EDTA, and preferably about 0.1% trypsin/1 mM EDTA) to producesingle cells, which are then inoculated onto fresh feeder cells. Suchsubculture is usually performed every 1 to 3 days, but the cells shouldbe monitored in the meantime, and any morphologically abnormal cellsthat are discovered should be discarded.

ES cells can be allowed to differentiate into various types of cells,such as those of the longus capitis muscle, visceral muscles, or cardiacmuscle, through monolayer culture to high density under suitableconditions, or through suspension culture until the formation of a cellmass (M. J. Evans & M. H. Kaufman, Nature, Vol. 292, 154 (1981); G. R.Martin, Proc. Natl. Acad. Sci. USA, Vol. 78, 7634 (1981); and T. C.Doetschman et al., Journal of Embryology and Experimental Morphology,Vol. 87, 27 (1985)). Cells with deficient expression of DNA of thepresent invention obtained upon the differentiation of the ES cells ofthe invention are useful for in vitro cytobiological analysis ofpolypeptides of the present invention and receptor proteins of theinvention.

Non-human mammals with deficient expression of DNA of the presentinvention can be distinguished from normal animals by assaying thelevels of mRNA in the animals in the usual manner and by indirectlycomparing the levels of expression.

Examples of such non-human mammals include those noted above.

Non-human mammals with deficient expression of DNA of the presentinvention can be produced by knocking out DNA of the invention throughhomologous recombination, where a targeting vector prepared as describedabove is introduced to mouse embryonic stem cells or mouse oocytes, andas a result of its introduction, the DNA sequence of the targetingvector with inactivated DNA of the invention replaces DNA of theinvention on the chromosomes of the mouse embryonic stem cells or mouseoocytes through genetic homologous recombination.

Cells in which DNA of the invention has been knocked out can bedetermined by Southern hybridization analysis with probe comprising aDNA sequence on or near DNA of the invention, or by PCR with primerscomprising the DNA sequence on the targeting vector and a DNA sequencein a nearby region other than the mouse DNA of the invention used in thetargeting vector. When non-human mammal embryonic stem cells are used, acell line in which DNA of the present invention has been inactivated byhomologous recombination can be cloned, the cells can be injected intonon-human mammal embryos or blastocysts at a suitable stage, such as the8-cell stage, and the resulting chimeric embryos can be transplanted tothe uterus of a surrogate non-human mammal. The resulting animal will bea chimeric animal comprising both cells with the normal DNA locus of thepresent invention and cells with the artificially mutated DNA locus ofthe present invention.

When some germ cells of the chimeric animal have the mutated DNA locusof the present invention, chimeric individuals can be mated with normalindividuals, and individuals in which all tissue comprises cells withthe artificially mutated DNA locus of the invention can be selected fromthe group of individuals resulting from the above mating, on the basisof coat color, for example. The resulting individuals are usuallycharacterized by deficient heterogeneous expression of polypeptides ofthe invention. The mating of individuals with deficient heterogeneousexpression of polypeptides of the invention or receptor proteins of theinvention can produce individuals with deficient homogeneous expressionof the polypeptides of the invention or receptor proteins of theinvention.

When oocytes are used, DNA solution can be injected by microinjectioninto the nucleus of the oocytes to produce transgenic non-human mammalswith the targeting vector introduced into the chromosomes, and thoseanimals with mutations in the DNA locus of the present inventionresulting from homologous recombination can be selected in comparison tothe above transgenic non-human mammals.

Individuals in which DNA of the present invention has been knocked outcan be mated to verify that the resulting individuals also have the DNAknocked out, and can be bred and raised under the usual breedingconditions.

The germ line should be obtained and maintained in the usual manner.Specifically, animals of both sexes conserving the inactivated DNA canbe mated to obtain homozygous animals with the inactivated DNA in bothchromosomes. The resulting homozygous animals can be efficientlyobtained when bred under conditions giving 1 normal individual andseveral homozygotes per dam. Heterozygous animals of both sexes can bemated to breed and raise heterozygous and homozygous animals with theinactivated DNA.

Non-human mammal embryonic stem cells in which DNA of the invention hasbeen inactivated are extremely useful for producing non-human mammalswith deficient expression of DNA of the invention.

Because non-human mammals with deficient expression of DNA of theinvention lack various types of physiological activity which can beinduced by polypeptides of the invention or receptor proteins of theinvention, such animals can serve as models of disease caused byinactivation of the physiological activity of polypeptides of theinvention or receptor proteins of the invention, and can thus be usefulto research the causes of such diseases and to study therapies for them.

The abbreviations for bases, amino acids, and the like in theSpecification and drawings are based on the abbreviations authorized bythe IUPAC-IUB Commission on Biochemical Nomenclature, or otherabbreviations commonly used in the art, examples of which are givenbelow. Unless otherwise indicated, amino acid optical isomers are the Lform.

DNA: deoxyribonucleic acid cDNA: complementary deoxyribonucleic acid A:adenine T: thymine G: guanine C: cytosine I: inosine R: adenine (A) orguanine (G) Y: thymine (T) or cytosine (C) M: adenine (A) or cytosine(C) K: guanine (G) or thymine (T) S: guanine (G) or cytosine (C) W:adenine (A) or thymine (T) B: guanine (G), guanine (G) or thymine (T) D:adenine (A), guanine (G) or thymine (T) V: adenine (A), guanine (G) orcytosine (C) N: adenine (A), guanine (G) , cytosine (C), or thymine (T)or another unknown base RNA: ribonucleic acid mRNA: messengerribonucleic acid dATP: deoxyadenosine triphosphate dTTP: deoxythymidinetriphosphate dGTP: deoxyguanosine triphosphate dCTP: deoxycytidinetriphosphate ATP: adenosine triphosphate EDTA: ethylenediaminetetraceticacid SDS: sodium dodecylsulfate BHA: benzhydrylamine pMBHA:p-methylbenzhydrylamine Tos: p-toluenesulfonyl Bzl: benzyl Bom:benzyloxymethyl Boc: t-butyloxycarbonyl DCM: dichloromethane HOBt:1-hydroxybenztriazole DCC: N,N′-dicyclohexylcarbodiimide TFA:trifluoroacetic acid DIEA: diisopropylethylamine Gly or G: glycine Alaor A: alanine Val or V: valine Leu or L: leucine Ile or I: isoleucineSer or S: serine Thr or T: threonine Cys or C: cystine Met or M:methionine Glu or E: glutamic acid Asp or D: aspartic acid Lys or K:lysine Arg or R: arginine His or H: histidine Phe or F: phenylalanineTyr or Y: tyrosine Trp or W: tryptophan Pro or P: proline Asn or N:asparagine Gin or Q: glutamine pGlu: pyroglutamic acid NMP: N-methylpyrrolidone Cl-Z: 2-chlorobenzyloxycarbonyl Br-Z:2-bromobenzyloxycarbonyl OcHex: cyclohexyl ester OBzl: benzyl ester Tos:p-toluenesulfonyl HOBt: 1-hydroxybenztriazole MeBzl: 4-methyl benzylBom: benzyloxymethyl DCC: N,N′-dichlorohexylcarbodiimide BHA:benzhydrylamine pMBHA: p-methylbenzhydrylamine CHO: formyl

The SEQ ID NOS. in the Sequence Listing of the present applicationindicate the following sequences.

[SEQ ID NO. 1]

base sequence of primer used in Example 1 below to clone ATTα

[SEQ ID NO. 2]

base sequence of primer used in Example 1 below to clone ATTα

[SEQ ID NO. 3]

amino acid sequence coding for ATTα

[SEQ ID NO. 4]

base sequence of DNA coding for ATTα

[SEQ ID NO. 5]

base sequence of DNA including base sequence of DNA coding for ATTαcloned in Example 1

[SEQ ID NO. 6]

amino acid sequence of secretion signal sequence of ATTα

[SEQ ID NO. 7]

amino acid sequence coding for ATT

[SEQ ID NO. 8]

base sequence of primer used in Example 2 below to clone ATTβ

[SEQ ID NO. 9]

base sequence of primer used in Examples 2 and 5 below to clone ATTβ

[SEQ ID NO. 10]

base sequence of primer used in Example 2 below to clone ATTβ

[SEQ ID NO. 11]

base sequence of primer used in Examples 2 and 5 below to clone ATTβ

[SEQ ID NO. 12]

base sequence of DNA including base sequence of DNA coding for ATTβcloned in Example 2

[SEQ ID NO. 13]

amino acid sequence coding for ATTβ

[SEQ ID NO. 14]

base sequence of DNA coding for ATTβ

[SEQ ID NO. 15]

base sequence of primer used in Example 2 below to clone ATTβ

[SEQ ID NO. 16]

base sequence of DNA including base sequence of DNA coding for ATTηcloned in Example 2

[SEQ ID NO. 17]

amino acid sequence coding for ATTshort1

[SEQ ID NO. 18]

base sequence of DNA including base sequence of DNA coding for ATT#21Fcloned in Example 5

[SEQ ID NO. 19]

insert sequence described in Example 5

[SEQ ID NO. 20]

amino acid sequence coding for ATT#21F

[SEQ ID NO. 21]

base sequence of DNA coding for ATT#21F

[SEQ ID NO. 22]

amino acid sequence coding for ATTshort2

[SEQ ID NO. 23]

amino acid sequence coding for 1 amino acid deletion at N terminal ofATTshort2 (C terminal amide synthesized in Example 8)

[SEQ ID NO. 24]

amino acid sequence coding for 2 amino acid deletion at N terminal ofATTshort2 (C terminal amide synthesized in Example 9)

[SEQ ID NO. 25]

amino acid sequence coding for 3 amino acid deletion at N terminal ofATTshort2 (C terminal amide synthesized in Example 10)

[SEQ ID NO. 26]

amino acid sequence coding for 4 amino acid deletion at N terminal ofATTshort2 (C terminal amide synthesized in Example 11)

[SEQ ID NO. 27]

amino acid sequence coding for peptide with N terminal Gln of peptiderepresented by SEQ ID NO. 26 converted to pyroglutamate (C terminalamide synthesized in Example 12)

[SEQ ID NO. 28]

amino acid sequence coding for peptide with Arg added to N terminal ofATTshort2 (C terminal amide synthesized in Example 13)

[SEQ ID NO. 29]

base sequence of DNA coding for ATT

[SEQ ID NO. 30]

base sequence of DNA coding for ATTshort1

[SEQ ID NO. 31]

base sequence of DNA coding for ATTshort2

[SEQ ID NO. 32]

amino acid sequence of novel motif present on C terminal of ATTshort2

[SEQ ID NO. 33]

base sequence of DNA coding for novel motif present on C terminal ofATTshort2

[SEQ ID NO. 34]

amino acid sequence coding for 1 amino acid deletion at N terminal ofATTshort1

[SEQ ID NO. 35]

amino acid sequence coding for 2 amino acid deletion at N terminal ofATTshort1

[SEQ ID NO. 36]

amino acid sequence coding for 3 amino acid deletion at N terminal ofATTshort1

[SEQ ID NO. 37]

amino acid sequence coding for 4 amino acid deletion at N terminal ofATTshort1

[SEQ ID NO. 38]

amino acid sequence coding for 5 amino acid deletion at N terminal ofATTshort1

[SEQ ID NO. 39]

amino acid sequence of 5 amino acids at C terminal of ATTshort2

[SEQ ID NO. 40]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 23

[SEQ ID NO. 41]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 24

[SEQ ID NO. 42]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 25

[SEQ ID NO. 43]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 26

[SEQ ID NO. 44]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 27

[SEQ ID NO. 45]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 28

[SEQ ID NO. 46]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 34

[SEQ ID NO. 47]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 35

[SEQ ID NO. 48]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 36

[SEQ ID NO. 49]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 37

[SEQ ID NO. 50]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 38

[SEQ ID NO. 51]

base sequence of DNA coding for peptide with amino acid sequencerepresented by SEQ ID NO. 39

[SEQ ID NO. 52]

partial base sequence of DNA coding for ATTαspecifically amplified inExample 6

[SEQ ID NO. 53]

base sequence of sense strand primer used to obtain human ATTβ inExample 14

[SEQ ID NO. 54]

base sequence of antisense strand primer used to obtain human ATTβ inExample 14

[SEQ ID NO. 55]

base sequence of DNA fragment containing DNA sequence coding for humanATTβ obtained in Example 14

[SEQ ID NO. 56]

base sequence of sense strand primer used to obtain ATT#21F in Example14

[SEQ ID NO. 57]

base sequence of antisense strand primer used to obtain ATT#21F inExample 14

[SEQ ID NO. 58]

base sequence of DNA fragment containing DNA sequence coding for humanATT#21F obtained in Example 14

[SEQ ID NO. 59]

base sequence of sense strand primer used to obtain human PPT-A inExample 14

[SEQ ID NO. 60]

base sequence of antisense strand primer used to obtain human PPT-A inExample 14

[SEQ ID NO. 61]

base sequence of sense strand primer used to obtain human PPT-A inExample 14

[SEQ ID NO. 62]

base sequence of antisense strand primer used to obtain human PPT-A inExample 14

[SEQ ID NO. 63]

amino acid sequence coding for human PPT-A obtained in Example 14

[SEQ ID NO. 64]

base sequence coding for amino acid sequence represented by SEQ ID NO.63

The transformant Escherichia coli DH5α/pTBN1 obtained in Example 1 is ondeposit under the accession number FERM BP-6959 at the NationalInstitute of Bioscience and Human Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan as of Dec. 6,1999, and is also on deposit under the accession number IFO 16337 at theInstitute of Fermentation, Osaka (IFO), 2-17-85 Juso-honmachi,Yodogawa-ku, Osaka-shi, Japan as of Nov. 16, 1999.

The transformant Escherichia coli DH5α/pTBN2 obtained in Example 2 is ondeposit under the accession number FERM BP-6960 at the NationalInstitute of Bioscience and Human Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan (Postal No.305-8566) as of Dec. 6, 1999, and is also on deposit under the accessionnumber IFO 16338 at the Institute of Fermentation, Osaka (IFO), 2-17-85Juso-honmachi, Yodogawa-ku, Osaka-shi, Japan as of Nov. 16, 1999.

The transformant Escherichia coli DH5α/pTBN6 obtained in Example 5 is ondeposit under the accession number FERM BP-7094 at the NationalInstitute of Bioscience and Human Technology (NIBH), Agency ofIndustrial Science and Technology, Ministry of International Trade andIndustry, 1-1-3 Higashi, Tsukuba-shi, Ibaraki-ken, Japan as of Mar. 16,2000, and is also on deposit under the accession number IFO 16382 at theInstitute of Fermentation, Osaka (IFO), 2-17-85 Juso-honmachi,Yodogawa-ku, Osaka-shi, Japan as of Feb. 24, 2000.

EXAMPLES

Working Examples and Experimental Examples are given below to illustratethe invention in further detail, but the present invention is notlimited by these examples. Gene manipulation using Escherichia coli wasmanaged in accordance with the procedures noted in Molecular Cloning bySambrook et al. as noted above.

Example 1 Cloning of cDNA Coding for Human ATT Precursor Protein (ATTα)

cDNA coding for ATTα was obtained by PCR in the following manner. A 20μL mixture was prepared, containing 25 pmol each of the oligo DNArepresented by SEQ ID NO. 1 as sense strand primer and the oligo DNArepresented by SEQ ID NO. 2 as antisense strand primer, 10 μL of PremixTagq™ (Ex Taq™ Version) (by Takara Shuzo) as template DNA, and 1 μL cDNAsolution per tissue from human fetal multiple tissue cDNA panels(Clontech). A thermal cycler (GeneAmp (registered trademark) PCR systemmodel 9700 (Perkin Elmer)) was used to run PCR by a program consistingof 1 minute at 94° C., followed by 5 cycles alternating between 10seconds at 94° C. and 3 minutes at 72° C., 5 cycles alternating between10 seconds at 94° C. and 3 minutes at 70° C., and 25 cycles alternatingbetween 10 seconds at 94° C. and 3 minutes at 68° C., and then extensionfor 5 minutes at 68° C. The reaction liquid was electrophoresed on 2.0%agarose gel, and subsequent ethidium bromide staining revealed a bandcorresponding to DNA amplified by PCR around the 0.4 kb position, asdetermined on the basis of the molecular marker in a reaction systemusing human fetal skeletal muscle cDNA as template DNA. A QIAquick GelExtraction kit (Qiagen) was used to collect the DNA fragments, pCR(registered trademark) 2.1-TOPO (Invitrogen) was used to sequence thebase sequence for TA cloning, and the above plasmid was introduced intoE. coli DH5α competent cells. Clones with plasmids having the exogenousDNA were selected from clones of ampicillin resistant transformantsappearing on LB agar medium containing ampicillin, and pTBN1 plasmid DNAwas prepared.

To sequence the base sequence of the DNA insert, the pTBN1 was used astemplate DNA, and two types (PRM-007 and PRM-008) of commerciallyavailable primer DNA (Toyo Boseki) were used as sequence primers. Thesequencing reaction was run with the aid of an ABI Prism (registeredtrademark) BigDye Terminator Cycle Sequencing FS Ready Reaction Kit(Perkin Elmer) according to the conditions in the accompanying protocol,and with the use of the thermal cycler (GeneAmp (registered trademark)PCR system model 9700 (Perkin Elmer)), and the reaction material wasanalyzed with a DNA Sequencer ABI Prism (registered trademark) 377(Perkin Elmer).

The pTBN1 contained a 373 base pair DNA fragment represented by SEQ IDNO. 5, which contained an open reading frame consisting of the basesequence of 204 bases represented by SEQ ID NO. 4, coding for a novelpolypeptide consisting of the 68 amino acids represented by SEQ ID NO. 3having no homology with any known proteins (FIG. 1). Sequences presentin the encoded polypeptide included a typical 16 amino acid residuesecretion signal represented by SEQ ID NO. 6, and a consensus sequence(Gly-Lys-Arg) which had undergone carboxyl terminal amidation bycarboxypeptidase and restriction cleavage by a processing enzyme(endoprotease) from the amino acid residue at position 62 to that atposition 64. In vivo, this restriction cleavage results in theproduction of the mature 45 amino acid residue peptide having theamidated carboxyl terminal sequence represented by SEQ ID NO. 7, forexample. Although this peptide had the amidated carboxyl terminalsequence Phe-Xaa-Gly-Leu-Met-NH₂ motif found in tachykinins, the primarystructure of the amino terminal side was completely different from knowntachykinins, and it was therefore designated ATT (atypical tachykinin).The precursor ATTα represented by SEQ ID NO. 3 was referred to as ATTα.

The transformant E. coli DH5α/pTBN1 was obtained when the plasmid pTBN1with the DNA coding for the ATTα precursor of the human ATT obtained inthis example was introduced into E. coli DH5α.

Example 2 Analysis of Full Length cDNA Coding for Human ATT PrecursorProtein (ATTβ)

The following is a summary of the analysis of the full length cDNAcoding for ATTβ by 5′ RACE (rapid amplification of cDNA end) and 3′RACE. The template DNA for the RACE PCR was Marathon™-Ready cDNA humanheart (Clontech) or Marathon™-Ready cDNA human fetal lung (Clontech).5′RACE was run using the oligo DNA represented by SEQ ID NO. 8 as theantisense strand primer, and 3′ RACE was run using the oligo DNArepresented by SEQ ID NO. 9 as the sense strand primer during theprimary PCR reaction based on the DNA sequence coding for ATTα; then,during nested PCR, 5′ RACE was run using the oligo DNA represented bySEQ ID NO. 10 as the antisense strand primer, and 3′ RACE was run usingthe oligo DNA represented by SEQ ID NO. 11 as the sense strand primer,giving the 5′ upstream sequence and 3′ downstream sequence, with thenested primers as starting points. Sequencing of the base sequences ofthe resulting double-stranded DNA revealed that both sequences of the 5′upstream and 3′ downstream sides were the same regardless of thetemplate DNA used. The full length 720 base pairs represented by SEQ IDNO. 12, including the poly(A)⁺ chain, were thus assumed to be the basesequence of the full length cDNA of the ATT precursor protein. The basesequence of this DNA includes an open reading frame, consisting of the228 bases represented by SEQ ID NO. 14, coding for the novel polypeptideconsisting of the 76 amino acids represented by SEQ ID NO. 13, with nohomology to any known proteins (FIG. 2). The encoded polypeptide sharedthe sequence from the amino acid terminal to the Gly at position 67 incommon with the ATTα obtained in Example 1. This protein was designatedATTβ because it had a precursor structure producing the mature peptideATT in the body in the same manner as ATTα. PCR cloning was done in thesame manner as in Example 1 to obtain the actual cDNA fragment codingfor ATTβ. Specifically, a 25 μL mixture was prepared, containing 25 pmoleach of the oligo DNA represented by SEQ ID NO. 1 as sense strand primerand the oligo DNA represented by SEQ ID NO. 15 as antisense strandprimer, 12.5 μL of Premix Taq™ (Ex Taq™ Version) (by Takara Shuzo) astemplate DNA, and 1 μL cDNA solution per tissue from human fetalmultiple tissue cDNA panels (Clontech). A thermal cycler (GeneAmp(registered trademark) PCR system model 9700 (Perkin Elmer)) was used torun PCR by a program consisting of 3 minutes at 94° C., followed by 5cycles alternating between 10 seconds at 94° C. and 3 minutes at 72° C.,5 cycles alternating between 10 seconds at 94° C. and 3 minutes at 70°C., and 25 cycles alternating between 10 seconds at 94° C. and 3 minutesat 68° C., and then extension for 5 minutes at 68° C. pTBN2 plasmid DNAwas obtained upon the same TA cloning procedure as in Example 1 for PCRproducts of a reaction system using template DNA comprising human fetalheart cDNA, where a DNA band had been detected from around the 0.4 kb to0.5 kb positions, as determined on the basis of the molecular marker,upon 2.0% agarose gel electrophoresis of the reaction liquid andsubsequent ethidium bromide staining. Sequencing of the base sequence ofthe DNA insert in the pTBN2 in the same manner as in Example 1 revealedthe predicted 469 base pair DNA fragment represented by SEQ ID NO. 16,which included the base sequence represented by SEQ ID NO. 14 coding forATTβ. cDNA fragments sharing the sequence coding for ATTβ were amplifiedfrom human heart or human fetal skeletal muscle as well as human fetallung cDNA by PCR under the same conditions. In PCR tests intended toamplify partial cDNA fragments of the coding region of ATTβ, the DNA wasamplified when using human adipose tissue and human pituitary cDNA astemplate.

cDNA was thus found to code for both ATTα and ATTβ, which are precursorsof the aforementioned ATT peptide, and comparison of the base sequencesindicated they were splice variants from the same gene.

The plasmid pTBN2 with DNA coding for the human ATT precursor proteinATTβ obtained in the example was introduced into E. coli DH5α, givingthe transformant E. coli DH5α/pTBN2.

Example 3 Preparation of Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met-NH₂

ATT was synthesized according to the following procedure (amidatedcarboxyl group of the C terminal methionine in SEQ ID NO. 17, referredto as “ATT(35-45)”).

A 0.5 mmol portion of commercially available p-methyl BHA resin (0.57mmole/g-resin) was introduced into the reaction tank of an ABI 430Apeptide synthesizer, and Boc-Met, Boc-Leu, Boc-Gly, Boc-Phe, Boc-Phe,Boc-Gln, Boc-Ser(Bzl), Boc-Ala, Boc-Lys(Cl-Z), Boc-Gly, and Boc-Thr(Bzl)were introduced, in that sequence, by Boc-strategy (NMP-HOBt) peptidesynthesis, giving a protected peptide resin. 0.14 g of this resin wasstirred for 60 minutes at 0° C. along with 1.5 mL p-cresol in 10 mLanhydrous hydrogen fluoride, the hydrogen fluoride was then distilledoff at reduced pressure, diethyl ether was added to the residue forfiltration, and the residue was extracted with aqueous acetic acid. Theextract was thoroughly concentrated, loaded on a Sephadex (registeredtrademark) G-25 column (2.0×80 cm) packed with 50% aqueous acetic acid,and eluted with the same solvent to collect the primary fractions, whichwere loaded on a reverse phase chromatogram (2.6×8.0 cm) packed withLiChroprep (registered trademark) RP-18, washed with 200 mL of 0.1%aqueous TFA, and eluted with a linear gradient using 300 mL of 10%aqueous acetonitrile containing 0.1% TFA and 300 mL of 40% aqueousacetonitrile containing 0.1% TFA, and the primary fractions werecollected and lyophilized, giving 26 mg white powder.

Mass analysis (M+H)⁺ 1185.6 (theoretical 1185.6); HPLC elution time:17.3 min;

Column Conditions

column: Wakosil 5C18T 4.6×100 mm

eluant: Liquid A: 0.1% aqueous TFA and Liquid B: acetonitrile containing0.1% TFA used for elution (25 min) with a linear gradient A/B: 95/5 to45/55

flow rate: 1.0 mL/min

Example 4Thr-Val-Ala-Gly-Asp-Gly-Gly-Glu-Glu-Gln-Thr-Leu-Ser-Thr-Glu-Ala-Glu-Thr-Trp-Glu-Gly-Ala-Gly-Pro-Ser-Ile-Gln-Leu-Gln-Leu-Gln-Glu-Val-Lys-Thr-Gly-Lys-Ala-Ser-Gln-Phe-Phe-Gly-Leu-Met-NH₂

ATT was synthesized according to the following procedure (amidatedcarboxyl group of the C terminal methionine in SEQ ID NO. 7, referred toas “ATT(1-45)”).

Protected peptide resin with the amino acid sequence of ATT(1-45) wasobtained by further adding Boc amino acids according to the desiredsequence to the amino terminal side of the protected peptide resinprepared up to Boc-Thr(Blz) in Example 1. This resin was treated withhydrogen fluoride and purified by column chromatography in the samemanner as in Example 1 to obtain ATT(1-45).

Mass analysis (M+H)⁺ 4727.6 (theoretical 4728.2); HPLC elution time:20.4 min.

Column Conditions

column: Wakosil 5C18T 4.6×100 mm

eluant: Liquid A: 0.1% aqueous TFA and Liquid B: acetonitrile containing0.1% TFA used for elution with a linear gradient (25 min) A/B: 95/5 to45/55

flow rate: 1.0 mL/min

Example 5 3′-RACE Analysis Using Human Fetal Skeletal Muscle cDNA

After Example 2, 3′-RACE analysis was performed using human fetalskeletal muscle cDNA. Marathon™-Ready cDNA human fetal skeletal muscle(Clontech) was used as the template DNA for RACE PCR. The reaction wascarried out in the same manner as in Example 2 using the oligo DNArepresented by SEQ ID NO. 9 as the sense strand primer in primary PCRand then the oligo DNA represented by SEQ ID NO. 11 as the sense strandprimer in nested PCR. The reaction liquid was electrophoresed on 2.0%agarose gel, and ethidium bromide staining revealed other RACE PCRproducts of a size greater than that of the DNA fragment of a lengthcorresponding to the reaction product obtained in Example 2. The PCRproducts were collected in the same manner as in Example 1 and TAcloned, and plasmids were then introduced into E. coli DH5α competentcells. Several clones with plasmids having the exogenous DNA fragmentsincorporated therein were selected from clones of ampicillin resistanttransformants appearing on LB agar medium containing ampicillin, plasmidDNA was prepared, and the base sequences of the inserted DNA fragmentswere sequenced. Plasmid pTBN6 corresponding to clone #21 was found toinclude the 539 bases represented by SEQ ID NO. 18, starting at thenested primer represented by SEQ ID NO. 11, as well as a DNA fragmentwith the polyA sequence added to the 3′ side. Comparison of this basesequence with that of the 3′-RACE PCR product obtained in Example 2revealed that this base sequence included the 93 base insert sequencerepresented by SEQ ID NO. 19, but the two base sequences were otherwisecompletely consistent with each other except for the length of the 3′end polyA sequence. In conjunction with the results of Example 2, it wasthus concluded that the gene for the ATT precursor protein included anovel splice variant producing the base sequence represented by SEQ IDNO. 21 coding for a novel polypeptide (ATT#21F) consisting of the 107amino acids represented by SEQ ID NO. 20. This novel polypeptide notonly had the precursor structure producing a peptide similar to themature peptide ATT produced by ATTα and ATTβ above, but the carboxylterminal side also included a consensus sequence (Gly-Lys-Arg) which hadundergone carboxyl terminal amidation by carboxypeptidase andrestriction cleavage by a processing enzyme (endopeptidase) from theamino acid residue at position 90 to that at position 92 in SEQ ID NO.20, and the sequence from positions 74 to 77 of SEQ ID NO. 20(Arg-Arg-Lys-Lys) was also a restriction cleavage motif resulting from aprocessing enzyme (endoprotease). In vivo, this restriction cleavageresults in the production of a novel sequence of 14 amino acid residueswith the amidated carboxyl terminal sequence represented by SEQ ID NO.22. The amidated carboxyl terminal motif of the peptide isPhe-Xaa-Gly-Leu-Leu-NH₂. One terminal amino acid (Leu/Met) is differentfrom the Phe-Xaa-Gly-Leu-Met-NH₂ motif of conventional tachykininfamily. This structure has not been found in any known naturallyoccurring proteins or peptides.

The pTBN6 plasmid analyzed in this example was introduced into E. coliDH5α, giving the transformant E. coli DH5α/pTBN6.

Example 6 Chromosome Mapping of Gene Coding for Human ATT PrecursorProteins

Radiation Hybrid analysis was employed in the following manner forchromosomal mapping of the gene coding for human ATT precursor proteins.PCR using the oligo DNA represented by SEQ ID NO. 11 as the sense strandprimer, the oligo DNA represented by SEQ ID NO. 2 as the antisensestrand primer, and human chromosomal DNA as template resulted in thespecific amplification of the 160 base pair DNA fragment represented bySEQ ID NO. 52, corresponding to a portion of the base sequence of theATTα above. PCR was carried out with the same combination of primers fora total of 83 DNA clones in Stanford G3 Radiation Hybrid Panels(Research Genetics), the reaction solutions were electrophoresed on 2%agarose gel, and the gels were stained with ethidium bromide to checkfor the presence or absence of amplified PCR reaction products of thesame size as above. The resulting patterns which appeared for theamplified products of each clone were sent to the SHGC RH Server at theStanford Human Genome Center to search for markers. Based on theresulting information, such as SHGC-52587, SHGC-2255, and SHGC-828 whichshowed up as hits in terms of neighboring markers, the locus of the genecoding for the polypeptide of the invention was mapped to the long arm(17q21) of human chromosome 17.

Example 7 Preparation ofLys-Lys-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂ (SEQ ID NO:22)

The following procedure was used to synthesize the polypeptiderepresented by SEQ ID NO. 22 obtained in Example 5, in which thecarboxyl group of the C terminal leucine was amidated.

A 0.5 mmole portion of commercially available p-methyl BHA resin (0.57mmole/g-resin) was introduced into the reaction tank of an ABI 430Apeptide synthesizer, and Boc-Leu, Boc-Leu, Boc-Gly, Boc-Gln, Boc-Phe,Boc-Thr(Bzl), Boc-His(Bom), Boc-Glu(OcHex), Boc-Leu, Boc-Gln,Boc-Tyr(Br-z), Boc-Ala, Boc-Lys(Cl-z), Boc-Lys(Cl-Z) were introduced, inthat sequence, by Boc-strategy (NMP-HOBt) peptide synthesis, giving aprotected peptide resin. The resin was stirred for 60 minutes at 0° C.along with p-cresol in anhydrous hydrogen fluoride, and the hydrogenfluoride was then distilled off at reduced pressure. Diethyl ether wasadded to the residue for filtration, the residue was extracted withaqueous acetic acid, the extract was thoroughly concentrated, loaded ona Sephadex™ G-25 column (2.0×80 cm) packed with 50% aqueous acetic acid,and eluted with the same solvent to collect the primary fractions, whichwere lyophilized. These were dissolved in 0.1% TFA aqueous solution andloaded on a reverse phase chromatogram (2.6×8.0 cm) packed withLiChroprep™ RP-18, washed with 200 mL of 0.1% aqueous TFA, and elutedwith a linear gradient using 300 mL of 10% aqueous acetonitrilecontaining 0.1% TFA and 300 mL of 40% aqueous acetonitrile containing0.1% TFA, and the primary fractions were collected and lyophilized,giving a white powder.

Mass analysis (M+H)⁺ 1674.9 theoretical 1674.9; HPLC elution time: 17.0min.

Column Conditions

column: Wakosil 5C18T 4.6×100 mm

eluant: Liquid A: 0.1% aqueous TFA and Liquid B: acetonitrile containing0.1% TFA used for elution (25 min) with a linear gradient A/B: 95/5 to45/55

flow rate: 1.0 mL/min

Example 8 Preparation ofLys-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂ (Amide ofPeptide Consisting of Amino Acid Sequence Represented by SEQ ID NO. 23)

The target white powder was obtained when resin was purified in the samemanner as in Example 7 before the introduction of the finalBoc-Lys(Cl-Z) in Example 7.

Mass analysis (M+H)⁺ theoretical 1546.8.

Example 9 Preparation ofAla-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂ (Amide of PeptideConsisting of Amino Acid Sequence Represented by SEQ ID NO. 24)

The target white powder was obtained when resin was purified in the samemanner as in Example 8 up to the introduction of the Boc-Ala in Example7.

Mass analysis (M+H)⁺ theoretical 1418.7.

Example 10 Preparation ofTyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂ (Amide of PeptideConsisting of Amino Acid Sequence Represented by SEQ ID NO. 25)

The target white powder was obtained when resin was purified in the samemanner as in Example 8 up to the introduction of the Boc-Tyr(Br-Z) inExample 7.

Mass analysis (M+H)⁺ theoretical 1347.7.

Example 11 Preparation of Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂(Amide of Peptide Consisting of Amino Acid Sequence Represented by SEQID NO. 26)

The target white powder was obtained when resin was purified in the samemanner as in Example 8 before the introduction of the Boc-Tyr(Br-Z) inExample 7.

Mass analysis (M+H)⁺ theoretical 1184.6; HPLC elution time: 16.3 min.

Column Conditions

column: Wakosil 5C18T 4.6×100 mm

eluant: Liquid A: 0.1% aqueous TFA and Liquid B: acetonitrile containing0.1% TFA used for elution (25 min) with a linear gradient A/B: 95/5 to45/55

flow rate: 1.0 mL/min

Example 12 Preparation of pGlu-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂(Amide of Peptide Consisting of Amino Acid Sequence Represented by SEQID NO. 27)

The target white powder was obtained when the same resin used in Example11 was treated with anhydrous hydrogen fluoride and then set aside for20 hours at a pH of 7.5, the conversion of the N terminal Gln to pGluwas checked by HPLC, and the product was purified, giving the targetwhite powder.

Mass analysis (M+H)⁺ theoretical 1167.6; HPLC elution time: 18.1 min.

Column Conditions

column: Wakosil 5C18T 4.6×100 mm

eluant: Liquid A: 0.1% aqueous TFA and Liquid B: acetonitrile containing0.1% TFA used for elution (25 min) with a linear gradient A/B: 95/5 to45/55

flow rate: 1.0 mL/min

Example 13 Preparation ofArg-Lys-Lys-Ala-Tyr-Gln-Leu-Glu-His-Thr-Phe-Gln-Gly-Leu-Leu-NH₂ (Amideof Peptide Consisting of Amino Acid Sequence Represented by SEQ ID NO.28)

The target white powder was obtained when resin was purified in the samemanner as in Example 7 after Boc-Arg(Tos) had also been introduced intothe protected peptide resin prepared in Example 7.

Mass analysis (M+H)⁺ theoretical 1831.0.

Experimental Example 1 Activity of ATT(35-45) (Polypeptide C TerminalAmide Consisting of Amino Acid Sequence Represented by SEQ ID NO. 17) onBlood Pressure in Anesthetized Rats

The activity of ATT(35-45) on blood pressure in anesthetized rats wasdetermined in the following manner. Male Wistar rats (weighing 300 to400 g, by CLEA Japan Inc.) were anesthetized with thiobutabarbitalsodium (100 mg/kg i.p.), a catheter (SP-55) for measuring blood pressureconnected to a transducer was inserted into the left carotid artery, anda catheter (SP-35) for intravenous administration was inserted into theleft femoral vein. The peptide was dissolved in physiological salinecontaining 0.05% BSA, and doses of 0.01, 0.1, and 1 nmol/kg wereadministered through the left femoral vein. The blood pressure wascontinuously recorded on a polygraph (NEC Sanei).

Results: The administration of ATT(35-45) in doses of 0.01, 0.1, and 1nmol/kg resulted in dose-dependent decreases in blood pressure in theanesthetized rats. The decrease in blood pressure by dose was 12.3±0.8mmHg (n=4), 21.5±1.8 mmHg (n=4), and 33.8±6.6 mmHg (n=4), respectively(mean±standard deviation).

Experimental Example 2 Contracting Activity of ATT(35-45) on Ginea PigIleum Specimens

The contracting activity of ATT(35-45) on guinea pig ileum specimens wasdetermined in the following manner. 13 to 15-week old male guinea pigs(std: Hartley) were exsanguinated, and the ileum was excised. The ileumwas cut to a length of 1.5 cm to prepare specimens. The specimens wereaerated with a gas mixture (95% O²-5% CO₂) while suspended in Tyrode'ssolution (137.9 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl₂, 0.5 mM MgCl₂, 1.1 mMNaH₂PO₄, 11.9 mM NaHCO₃, and 5.6 mM glucose) maintained at 37° C., andthe isotonic contractions under a 0.5 g load were recorded. The peptidewas administered cumulatively in the organ bath.

Results: The administration of 10⁻¹⁰ M to 3×10⁻⁷ M ATT(35-45) resultedin concentration-dependent contraction of the guinea pig ileumspecimens. The maximum concentration was about 90% of the contractioninduced with 10⁻⁶ M acetylcholine. The EC₅₀ of the ATT(35-45) was 3.5 nM(n=6) (FIG. 3).

Example 14 Construction of Expression Vector

DNA fragments coding for the human ATT precursor protein (ATTβ) obtainedin Example 2 were obtained by PCR in the following manner. That is, a 50μL mixture was prepared, containing 20 pmol each of the synthetic oligoDNA represented by SEQ ID NO. 53 as the sense strand primer and thesynthetic oligo DNA represented by SEQ ID NO. 54 as the antisense strandprimer, 5 μL of 10× Advantage (registered trademark) 2×PCR buffer(Clontech), 1 μL 50×dNTP mix (Clontech), 1 μL of 50× Advantage 2Polymerase Mix (Clontech), and 1 ng of the pTBN2 plasmid obtained inExample 2 as the template DNA. A thermal cycler (GeneAmp (registeredtrademark) PCR system model 9700 (Applied Biosystems) was used to runPCR by a program consisting of 1 minute at 95° C., followed by 25 cyclesalternating between 10 seconds at 95° C. and 1 minute at 74° C., andthen extension for 7 minutes at 74° C. The resulting 0.25 kb fragmentswere subcloned to the pCR2.1-TOPO (Invitrogen) vector, and the plasmidwas introduced to E. coli JM109 (Takara Shuzo) competent cells. Cloneswith the plasmid having the exogenous DNA fragment incorporated thereinwere selected from colonies of ampicillin resistant transformants whichappeared on LB agar medium containing ampicillin to preparepCR2.1-TOPO/ATTβ plasmid DNA. The plasmid was double digested with therestriction enzymes EcoRI and SalI (Takara Shuzo). Then, 0.25 kb genefragments containing the ATTβ were separated and collected byelectrophoresis on agarose gel, and were purified using a QIAquick GelExtraction kit (Qiagen). The resulting fragments were subcloned to thepCAN618 vector, and the plasmid was introduced into E. coli JM109(Takara Shuzo) competent cells. Clones with the plasmid having theexogenous DNA fragment incorporated therein were selected from coloniesof ampicillin resistant transformants which appeared on LB agar mediumcontaining ampicillin to prepare pCAN-ATTβ plasmid DNA. The pCAN-ATTβcontained the 250 base pair DNA fragment represented by SEQ ID NO. 55coding for human ATTβ consisting of the 76 amino acids represented bySEQ ID NO. 13.

The DNA fragment coding for human ATT#21F determined in Example 5 wasobtained by PCR in the following manner. That is, a 50 μL mixture wasprepared, containing 20 pmol each of the synthetic oligo DNA representedby SEQ ID NO. 56 as the sense strand primer and the synthetic oligo DNArepresented by SEQ ID NO. 57 as the antisense strand primer, 5 μL of10×Advantage (registered trademark) 2×PCR buffer (Clontech), 1 μL50×dNTP mix (Clontech), 1 μL of 50× Advantage 2 Polymerase Mix(Clontech), and 5 μL DNA solution containing the sequence coding forhuman ATT#21F represented by SEQ ID NO. 21 as the template DNA. Athermal cycler (GeneAmp (registered trademark) PCR system model 9700(Applied Biosystems) was used to run PCR by a program consisting of 1minute at 95° C., followed by 20 cycles alternating between 5 seconds at95° C. and 10 seconds at 68° C., and then extension for 1 minute at 68°C. The reaction solution was double digested with the restrictionenzymes EcoRI and SalI (Takara Shuzo), and the short DNA fragmentsproduced by digestion with the restriction enzymes and unreacted primerswere removed using a QIAquick PCR Purification kit (Qiagen). Theresulting 0.34 kb fragments were subcloned to the pCAN618 vector, andthe plasmid was introduced into E. coli Epicurian Coli (registeredtrademark) XL10-Gold (registered trademark) line (Stratagene) competentcells. Clones with the plasmid having the exogenous DNA fragmentincorporated therein were selected from colonies of ampicillin resistanttransformants which appeared on LB agar medium containing ampicillin toprepare pCAN-ATT#21F plasmid DNA. The pCAN-ATT#21F contained a 340 basepair DNA fragment represented by SEQ ID NO. 58 coding for human ATT#21Fconsisting of the 107 amino acids represented by SEQ ID NO. 20.

A human PPT-A (substance P/neurokinin A precursor) expression vector wasprepared in the following manner for use in a comparative test. HumanPPT-A cDNA fragments were first isolated. That is, a 50 μL mixture wasprepared, containing 20 pmol each of the synthetic oligo DNA representedby SEQ ID NO. 59 as the sense strand primer and the synthetic oligo DNArepresented by SEQ ID NO. 60 as the antisense strand primer, 5 μL of 10×Advantage (registered trademark) 2×PCR buffer (Clontech), 1 μL 50×dNTPmix (Clontech), 1 μL of 50× Advantage 2 Polymerase Mix (Clontech), and 5μL Human MTC panel Brain solution as the template DNA. A thermal cycler(GeneAmp (registered trademark) PCR system model 9700 (AppliedBiosystems) was used to run PCR by a program consisting of 1 minute at96° C., followed by 25 cycles alternating between 10 seconds at 96° C.,5 seconds at 54° C., and 30 seconds at 72° C., and then extension for 2minutes at 72° C. The unreacted primers were removed from the reactionsolution using a QIAquick PCR Purification kit (Qiagen). The resultingDNA fragments were subcloned to the pCR2.1-TOPO (Invitrogen) vector, andthe plasmid was introduced into E. coli Epicurian Coli (registeredtrademark) XL10-Gold (registered trademark) line (Stratagene) competentcells. Clones with the plasmid having the exogenous DNA fragmentincorporated therein were selected from colonies of ampicillin resistanttransformants which appeared on LB agar medium containing ampicillin toprepare pCR2.1-TOPO/PPT-A plasmid DNA.

The DNA fragment coding for human PPT-A was then obtained by PCR in thefollowing manner. That is, a 50 μL mixture was prepared, containing 20pmol each of the synthetic oligo DNA represented by SEQ ID NO. 61 as thesense strand primer and the synthetic oligo DNA represented by SEQ IDNO. 62 as the antisense strand primer, 5 μL of 10× Advantage (registeredtrademark) 2×PCR buffer (Clontech), 1 μL 50×dNTP mix (Clontech), 1 μL of50× Advantage 2 Polymerase Mix (Clontech), and 1 ng pCR2.1-TOPO/PPT-Aplasmid as the template DNA. A thermal cycler (GeneAmp (registeredtrademark) PCR system model 9700 (Applied Biosystems) was used to runPCR by a program consisting of 1 minute at 96° C., followed by 20 cyclesalternating between 5 seconds at 96° C., 5 seconds at 66° C., and 30seconds at 72° C., and then extension for 1 minute at 72° C. Thereaction solution was double digested with the restriction enzymes EcoRIand SalI (Takara Shuzo), and the short DNA fragments produced bydigestion with the restriction enzymes and unreacted primers wereremoved using a QIAquick PCR Purification kit (Qiagen). The resulting0.4 kb fragments were subcloned to the pCAN618 vector, and the plasmidwas introduced into E. coli Epicurian Coli (registered trademark)XL10-Gold (registered trademark) line (Stratagene) competent cells.Clones with the plasmid having the exogenous DNA fragment incorporatedtherein were selected from colonies of ampicillin resistanttransformants which appeared on LB agar medium containing ampicillin toprepare PCAN-PPTA plasmid DNA. The PCAN-PPTA contained a 409 base pairDNA fragment represented by SEQ ID NO. 64 coding for human PPT-Aconsisting of the 129 amino acids represented by SEQ ID NO. 63.

Example 15 Introduction of Expression Vector to AtT-20 Cells andSecretion of Gene Product

To check the biosynthesis of the peptide from the ATT polypeptideprecursors, mouse pituitary AtT-20 cells were used to express the ATTpolypeptide precursors, and the following method was used to determinewhether or not the polypeptide was secreted in culture media. Petridishes 6 cm in diameter were inoculated with AtT-20 cells in aproportion of 2.5×10⁶ cells/dish on the day before transfection of theexpression vector, and were incubated for 24 hours in CO₂ in DMEM mediumsupplemented with 10% FBS (JRH) (by GibcoBRL). The pCAN-ATTβ,pCAN-ATT#21F, and pCAN-PPTA vectors expressing ATTβ, ATT#21F, and PPT-A,respectively, were transfected using Effectene (Qiagen) in a proportionof 1 μg/dish, respectively. After 24 hours, the medium was replaced withselection medium containing 500 μg/mL Geneticin. The medium was replacedabout every 5 days during 3 weeks of continuous culture, givingtransformants. After continued culture on selection medium, the cellswere replaced with serum-free medium, and the culture supernatant wasrecovered after another 2 days. The polypeptide in the resultingsupernatant was detected using a commercially available EIA kit,Substance P Enzyme Immunoassay Kit (Cayman Cat #583751). It waspreviously ascertained that the peptides obtained in Examples 3 and 4could be detected with the same degree of high sensitivity as SubstanceP when using the Substance P Enzyme Immunoassay Kit. The Substance PEnzyme Immunoassay Kit detected the secretion of peptides in the culturesupernatant of AtT-20 cells having expression vectors pCAN-ATTβ,pCAN-ATT#21F, and pCAN-PPTA. The biosynthesis of the peptide throughprocessing from ATT polypeptide precursors was thus confirmed.

Example 16 Preparation of Immunogen Containing Peptide Represented bySEQ ID NO. 22 in Example 5, and Immunization

Conjugates of hemocyanin (KLH) and the novel peptide obtained in Example7 were prepared for use as immunogen. That is, 10 mg KLH and 4.5 mg ofthe aforementioned peptide were dissolved and mixed in phosphate buffer(pH 6.5); glutaraldehyde was added; and a reaction was conducted for 1hour at ambient temperature. Following the reaction, the product wasdialyzed for 2 days at 4° C. against PBS, and the glutaraldehyde-freeproduct was used as immunogen (antigen protein).

For primary immunization, the antigen protein was subcutaneouslyadministered to rabbits (Japanese White) in an amount of 1 mg per animalin the form of an emulsion with FCA (Freund's Complete Adjuvant).Boosters were given every 2 weeks for a total of 4 times. For the secondand subsequent times, the dosage of antigen protein was 0.5 mg peranimal, given by subcutaneous injection in the form of an emulsion withFICA (Freund's Incomplete Adjuvant). One week following the finalbooster, blood was drawn from the auricular vein, and serum fractionswere obtained in the usual manner.

Example 17 Preparation of Western Horseradish Peroxidase (HRP) LabeledPeptide

The novel peptide obtained in Example 7 was cross-linked with HRP (forenzyme immunoassay, by Boehringer Mannheim) to produce labels for enzymeimmunoassay (EIA). That is, 8 mg HRP was dissolved in 0.95 mL of 0.02 Mphosphate buffer (pH 6.8), which was mixed with 0.05 mL DMF solutioncontaining 30-fold (molar) SPDP relative to the HRP, a reaction wasbrought about for 60 minutes at ambient temperature, 0.3 mL of 0.1 Macetic acid buffer (pH 4.5) containing 40 mM DTT was then added, and areaction was brought about for another 30 minutes at ambienttemperature. Fractions were obtained on a Sephadex G-25 columnequilibrated with 0.1 M phosphate buffer (pH 6.0) containing 2 mM EDTA,giving HRP with SH groups.

1.5 mg of the aforementioned peptide was meanwhile dissolved in 0.5 mLof 0.1 M phosphate buffer (pH 6.8) and mixed with 0.05 mL of DMFsolution containing GMBS in an equimolar amount relative to the peptide.A reaction was conducted for 60 minutes at ambient temperature, andfractions were then obtained on a Sephadex G-25 column equilibrated with0.1 M phosphate buffer (pH 6.8), giving peptides having maleimide groupsincorporated therein.

The HRP having the SH groups and the peptide having the maleimide groupswere then mixed in a molar ratio of 1:3 and allowed to react overnightat 4° C., and fractions were obtained on an Ultrogel AcA44 columnequilibrated with 0.1 M phosphate buffer (pH 6.5), giving HRP-labeledpeptides. The resulting labeled peptides were suitable as labels forenzyme immunoassay (EIA).

INDUSTRIAL APPLICABILITY

The present invention provides novel tachykinin polypeptides (referredto as ATT, ATT polypeptides, ATTshort1, or ATTshort2), their precursors(designated ATTα, ATTβ, or ATT#21F), and polynucleotides coding forthem. The invention also provides recombinant vectors containing suchpolynucleotides, transformants containing such vectors, and transgenicanimals having genes containing such polynucleotides. The inventionfurthermore provides methods for producing such polypeptides, antibodiesagainst such polypeptides, agonists and antagonists, and methods foridentifying them. The invention additionally provides pharmaceuticalcompositions comprising such polypeptides, polynucleotides, antagonists,antibodies, and receptors, and methods for treating or preventingdisease.

1. An isolated polypeptide, or amide or ester, or salts thereof, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:
 17. 2. The amide or salt thereof, of the polypeptide of claim
 1. 3. An isolated polypeptide according to claim 1, wherein the C terminal carboxyl group is amidated.
 4. A kit comprising the polypeptide, or amide or ester, or salts thereof, according to claim
 1. 5. A pharmaceutical composition comprising the polypeptide, or amide or ester thereof, or salts thereof, according to claim 1, and a pharmaceutically acceptable carrier, excipient or diluent.
 6. A method of making a pharmaceutical composition comprising adding a pharmaceutically acceptable carrier, excipient or diluent to the polypeptide of claim
 1. 7. A method for treating hypertension, said method comprising administering the polypeptide or amide or ester thereof, or their salts according to claim 1 to mammals in need thereof. 